Relationship of Chlorophyll Biosynthetic Heterogeneity to Photosynthetic Efficiency
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  • XIV. Relationship of Chlorophyll Biosynthetic Heterogeneity to Photosynthetic Efficiency

  • A. Prologue

  • B. Relatioship of Agricultural Productivity to Photosynthetic Effiviency

  • 1. The Primary photochemical acts of phtosystem I (PSI) and PSII

  • 2. Conversion of CO2 into Carbohydrates

  • 3. Theoretical maximal energy conversion efficiency of the phosynthetic electron transport system (PETS) of green plants

  • 4. Actual energy conversion efficiency of the PETS of green plants under field conditions

  • C. Molecular Basis of the Discrepancy between the Theoretical and Maximal Efficiencies of the PETS

  • 1. Contribution of extrinsic PETS parameters to photosynthetic efficiency

  • 2. Contribution of intrinsic PETS parameters to photosynthetic efficiency

  • D. Correction of the Antenna/Photosystem Chlorophyll Mismatch

  • E. What Kind of Scientific Knowledge is needed to Bioengineer a Reduction in Photosynthetic Unit (PSU) Size

  • 1. State of the art in our understanding of chlorophyll biosynthesis

  • a. Single-branched chlorophyll biosynthetic pathway (SBP)

  • b. Multibranched-branched chlorophyll biosynthetic pathway (MBP)

  • 2. Thylakoid-apoprotein biosynthesis

  • 3. Assembly of chlorophyll-protein complexes

  • a. Single-branched chlorophyll biosynthetic pathway-single location model (SBP-SLM)

  • b. Single-branched chlorophyll biosynthetic pathway-multilocation location model (SBP-MLM)

  • c. Multibranched-branched chlorophyll biosynthetic pathway-sublocation location model (MBP-SubLM)

  • 4. Which model is favored by experimental evidence

  • a. Incompatibility of the SBP-SLM with experimental data

  • b. Incompatibility of the SBP-MLM with experimental data

  • c. Compatibility of the MBP-SubLM with experimental data

  • F. Epilogue

  • C. References


    A. Prologue

    The world population of about 6 billion is expected to increase to 9 billion by the year 2030. It may reach 18 billion by the end of the century. Worldwide, there has been a progressive decline in cereal yield, and at present, the annual rate of yield increase is below the rate of population increase. Since it will be difficult to increase the land area under cultivation without serious environmental consequences, the increased demand for food and fiber will have to be met by higher agricultural plant productivity. Plant productivity depends in turn on photosynthetic efficiency. We have reason to believe that agricultural productivity can be significantly increased by alteration of the photosynthetic unit size. On the basis of recent advances in the understanding of the chemistry and biochemistry of the greening process and significant advances in molecular biology, we believe that alteration of the PSU size has become a realistic possibility.

    Life in the biosphere is carbon based. All molecules needed for life are made up of a carbon skeleton which is complemented by organic elements such as O, H, N, and inorganic elements such as K, P, Ca, Fe, etc. Carbon, O and H of organic compounds originate in CO2 and H2O. The carbon skeleton is assembled via the process of photosynthesis which essentially converts solar energy into chemical energy. Nitrogen originates in NH3 and inorganic elements originate in the rocks of the biosphere and are incorporated into the carbon skeleton via enzymatic reactions. Chemical energy consists of the covalent bond energy embedded into the carbon-carbon skeleton as well as the high energy bonds of ATP and NADPH which are formed during the process of photosynthesis.

    The carbon cycle essentially describes how photosynthesis supports organic life in the biosphere. The carbon skeleton formed via the process of photosynthesis is converted into the simple and complex food consumed by organic life. The needed energy for enzymatic interconversions and biosynthetic processes is provided by ATP and NADPH. The organic matter of dead biota is converted in turn into CO2, H2O, and inorganic elements by bacterial activity, then the cycle repeats itself all over again.

    At issue then, is whether agricultural productivity at today's levels of photosynthetic efficiency is efficient enough to feed a growing world population. Indeed the world population of about 6 billion is expected to increase to 9 billion by the year 2030 and may easily reach 18 billion by the end of the twenty first century.

    B. Relationship of Agricultural Productivity to Photosynthetic Effiviency

    Since plants form food by conversion of solar energy, CO2, and H2O into chemical energy via the process of photosynthesis, it ensues that agricultural productivity depends in turn upon photosynthetic efficiency. Let us therefore briefly dissect the components of photosynthetic efficiency.

    Photosynthetic efficiency is controlled by intrinsic and extrinsic factors (1). Extrinsic factors include the availability of water, CO2, inorganic nutrients, ambient temperature, and the metabolic and developmental state of the plant. The most important intrinsic factor is the efficiency of the photosynthetic electron transport system (PETS). The latter is driven by two photochemical reactions that take place in membrane-bound photosystem I (PSI), and PSII chlorophyll (Chl)-protein complexes.

    1. The Primary Photochemical Acts of Photosystem I (PSI) and PSII

    Conversion of solar energy into chemical energy is the results of two photochemical acts that take place in PSI and PSII. The primary photochemical act of PSII is initiated by the absorption of light by antenna Chl a and b. The absorbed photons are conveyed to special Chls in the PSII reaction center. There, the light energy is used to generate a strong oxidant Z+ which liberates oxygen from water. It also generates a weak reductant Q- which together with plastoquinone electron acceptor pools serve as temporary storage of the electrons extracted from water.The primary photoact of PSI is also initiated by the absorption of light by antenna Chl a and b, and in this case too, the absorbed photons are conveyed to special Chls in the PSI reaction center. There the light energy generates a weak oxidant P700* which receives electrons from the plastoquinone pools via cytochrome f and plastocyanin. It also generates a strong reductant A0 which donates electrons to NADP+ via a series of electron carriers and converts it to NADPH. The photochemical acts of PSII and PSI, and the flow of electrons between PSII and PSI are depicted in Fig. 1.
    Zscheme090606.jpg - 307626 Bytes
    Fig. 1. The Z scheme for electron transport in oxygenic photosynthesis (Reproduced from Ref 2.

    2. Conversion of CO2 into Carbohydrates

    During electron and proton flow, energy rich ATP and NADPH are formed. The energy of NAPDH and ATP is used for the enzymic conversion of CO2 into carbohydrates which are in turn converted into a variety of organic molecules. In summary the efficiency of food formation by green plants depends to a great extent on the efficiency of NADPH and ATP formation which depends in turn on the efficiency of the PETS.

    The rest of this chapter will therefore be devoted to a discussion of the efficiency of the PETS and possible alterations in the circuitry of the chloroplast that may lead to a higher efficiency of the PETS and higher plant productivity under field conditions.

    3. Theoretical Maximal Energy Conversion Efficiency of the PETS of Green Plants

    This discussion is essentially extracted from a 1975 RANN report (Lien and San Pietro, 1975).

    At the maximal quantum efficiency of one, two photons are required to move one electron across the potential difference of about 1.25 V between Z+ and A0. The maximal efficiency of the photochemical reactions leading to the formation of Z+ and A0 is given by
    E = 1.25 eV/ 2 h? (Eq 1)
    Where,
    E = efficiency of PETS
    eV = Energy units in electron volts
    h? = Energy of the absorbed photon in eV
    Since the red 680 nm photons absorbed by PSI and PSII have an energy of 1.83 eV, it ensues from Eq 1 that
    E = 1.25 eV/2*1.83 eV = 0.34 eV (Eq 2)
    Therefore the absolute maximal efficiency of the PETS under red light is
    (0.34 eV/1.25 eV)*100 = 27% (Eq 3)

    Under natural white light, although the Chl concentration in photosynthetic membranes is high enough to result in near total absorption of all incident photosynthetically active photons between 400 and 700 nm, under normal weather conditions, these photons represent only about 44.5 % of the total incident solar radiation. Therefore under these circumstances, the possible overall maximal energy conversion efficiency amounts to:
    (27%*44.5%)/100 = 12% (Eq 4)

    4. Actual Energy Conversion Efficiency of the PETS of Green Plants Under Field Conditions

    Under field conditions however, the average net photosynthetic efficiency results in a net agricultural productivity in the range of 2-8 tons of dry organic matter per acre per year (1). This corresponds to a solar conversion efficiency of 0.1 to 0.4% of the total average incident radiation. Therefore the discrepancy between the 12% maximal theoretical efficiency of the PETS, and the agricultural photosynthetic efficiency observed under field conditions ranges from
    (12%/0.4%)*100 = 3000%, (Eq 5) to
    (12%/0.1%)*100 = 12000% (Eq 6)

    C. Molecular Basis of the Discrepancy Between the Theoretical Maximal Efficiency of the PETS and the Actual Solar Conversion Efficiency of Photosynthesis Under Field Conditions

    The discrepancy between the 12% theoretical maximal efficiency of the PETS and the actual 0.1-0.4% solar conversion efficiency of photosynthesis observed under field conditions can be attributed to
    (a) factors extrinsic to the PETS, and
    (b) to intrinsic rate limitations of the PETS (Lien and San Pietro, 1975).

    1. Contribution of Extrinsic PETS Parameters to the Discrepancy Between the Theoretical Photosynthetic Efficiency of 12%and the Actual Photosynthetic Field Efficiency of 0.1-0.4%

    Photosynthetic efficiency under field conditions is directly or indirectly affected by extrinsic factors such as ambient weather conditions, availability of water, CO2, and inorganic nutrients, as well as the metabolic and developmental state of the plant. Some of those factors are under human control while others are not. They do contribute nevertheless, to the variation in photosynthetic efficiency under field conditions. The rest of this discussion will focus upon the impact of intrinsic factors that affect the PETS and photosynthetic efficiency.

    2. Contribution of Intrinsic PETS Parameters to the Discrepancy between the Theoretical Photosynthetic Efficiency of 12% and the Actual Photosynthetic Field Efficiency of 0.1-0.4%

    The 12% theoretical efficiency of the PETS assumes that under natural conditions, PSI and PSII operate at a maximal quantum efficiency of ONE. In other words, it is assumed that every absorbed photon is completely converted into energy without losses (Lien and San Pietro, 1975).

    Using the conventional figure of 200 light harvesting Chl molecules per reaction center (RC) per PS, i.e. for a photosynthetic unit (PSU) size of 200 per PS, under the moderate light intensities of a shady sky (about 1/10 of full sunlight), each RC would receive about 200 photons per second (s) ((Lien and San Pietro, 1975). In other words, each RC would receive about 200 hits or excitons per s. Under these conditions, in order to maintain a quantum efficiency of ONE, the slowest dark reaction of the entire PS must have a turnover rate of 200 per s (Lien and San Pietro, 1975).

    Under full sunlight, which is about 10 fold higher than in the shade, the turnover rate of the limiting dark-reaction should be 200*10 = 2000 per s. This turnover rate corresponds to a rate of O2 evolution of about 9000 ?moles of O2 per mg Chl per hour (h). Yet, the maximal rate of O2 evolution observed during a Hill reaction, which results in the oxidation of H2O and the release of O2, under saturating light intensities, and other optimal conditions, rarely exceeds 5-10% of the above value. In other words, it is equal to the optimal rate of O2 evolution observed in the shade (Lien and San Pietro, 1975).

    Furthermore extensive kinetic studies have demonstrated that the rate limiting steps of the PETS do not reside in the initial photochemical reactions that take place in the RC, but reside within the redox-carriers, i. e. the electron transport chains connecting PSII to PSI. The discrepancy between the capacity of the photon gathering apparatus, i. e. the antenna Chl-protein complexes and the capacity of the rate-limiting dark reactions has been named the antenna/PS Chl mismatch (Lien and San Pietro, 1975). Correction of the Antenna/PS Chl Mismatch

    The first and most important effect of the antenna/PS Chl mismatch is one of reduced quantum conversion efficiencies at light intensities above shade levels. The second effect relates to the photodestructive effects of the excess photons collected by antenna Chl but not used in the initial photochemical acts. The energy of these unused photons leads to serious photodestruction of the PETS which must be repaired at a cost (Lien and San Pietro, 1975).

    D. Correction of the Antenna/Photosystem Chlorophyll Mismatch

    Early on, the possible correction of the antenna/PS mismatch attracted the interest and curiosity of the photosynthesis community. It was suggested that one way of correcting the mismatch was by reducing the size of the PSU, which may be achieved by growing plants with chloroplasts having less antenna and more RC Chl per unit thylakoid area (Lien and San Pietro, 1975). Research performed in the early 1970s failed however in its effort to alter significantly the PSU size in algal cell cultures (Lien and San Pietro, 1975). Now, on the basis of deeper understanding of the chemistry and biochemistry of the greening process, which was achieved during the past 30 years, we have reason to believe that alteration of the PSU has become a realistic possibility.

    E. What Kind of Scientific Knowledge is Needed to Bioengineer a Reduction in PSU size

    Thorough and integrated anabolic and catabolic knowledge in the following fields of research is needed for successful research aimed at the bioengineering of a reduced PSU size namely:
    (a) Chl,
    (b) lipid,
    (c) carotenoid,
    (d) plastoquinone,
    (e) chloroplast apoprotein, and
    (f) assembly of pigment-protein complexes.
    Because of space limitations, the remainder of this discussion will focus on the Chl, and apoprotein components of chloroplasts as well as on the assembly of Chl-protein complexes.

    1.State of the Art in our Understanding of Chl Biosynthesis

    During the past 30 years, it has become apparent that contrary to previous beliefs, the Chl biosynthetic pathway, is not a simple single-branched pathway, but a complex multibranched pathway that consist of about 15 carboxylic and two fully esterified biosynthetic routes (Rebeiz et al, 2002). The single and multibranched carboxylic pathways are briefly discussed below.

    a.The Single-Branched Chl Biosynthetic Pathway Does Not Account for the Formation of All the Chl in Green Plants

    4SBPatway2B&W.jpg - 10329 Bytes

    Fig. 2. The single-branched Chl biosynthetic pathway.(reproduced from Rebeiz et al, 1994)

    The single-branched Chl biosynthetic pathway is depicted in Fig. 2. It consists of a linear sequence of biochemical reactions which convert divinyl (DV) protoporphyrin IX (Proto) to monovinyl (MV) Chl a via DV Mg-Proto, DV Mg-Proto monomethyl ester (Mpe), DV protochlorophyllide a (Pchlide a), MV Pchlide a, and MV Chlorophyllide a (Chlide a). The salient features of this pathway are (a) the assumption that DV Pchlide a does not accumulate in higher plants, but is a transient metabolite which is rapidly converted to MV Chl a via MV Pchlide a, and (b) that the formation and accumulation of MV tetrapyrroles between Proto and Mpe and DV tetrapyrroles between Pchlide a and Chl a does not take place (Rebeiz et al, 1994). All in all, experimental evidence gathered over the past 26 years indicates that only a small fraction of the total Chl of green plants is formed via this pathway (Rebeiz et al, 1994, 2002)

    b. The Chl of Green Plants is Formed via a Multibranched Biosynthetic Pathway

    Since the 1963 seminal review of Smith and French (Smith and French, 1963), our understanding of the Chl biosynthetic pathway has changed dramatically. Several factors have contributed to this phenomenon, among which: (a) development of systems capable of Chl and thylakoid membrane biosynthesis in organello and in vitro, (Rebeiz and Castelfranco, 1971a, 1971b, Daniell and Rebeiz, 1982a, 1982b, Rebeiz et al, 1984, Kolossov et al, 1999), (b) powerful analytical techniques that allowed the qualitative and quantitative determination of various intermediates of the pathway (Rebeiz, 2002), (c) recognition that the greening process proceeds differently in etiolated and green tissues, in darkness and in the light, ((Carey et al, 1985), and in plants belonging to different greening groups; Ioannides et al, 1994; Abd-El-Magid et al, 1997), and (d) recognition of the probability that the structural and functional complexity of thylakoid membranes is rooted in a multibranched, heterogeneous Chl biosynthetic pathway (Rebeiz et al, 1999).

    It was proposed that Chlorophyll biosynthetic heterogeneity referred either (a) to spatial biosynthetic heterogeneity, (b) to chemical biosynthetic heterogeneity, or (c) to a combination of spatial and chemical biosynthetic heterogeneities (Rebeiz et al, 2002). Spatial biosynthetic heterogeneity was defined as the biosynthesis of an anabolic tetrapyrrole or end product by identical sets of enzymes, at several different locations of the thylakoid membrane. On the other hand, chemical biosynthetic heterogeneity was defined as the biosynthesis of an anabolic tetrapyrrole or end product at several different locations of the thylakoid membrane, via different biosynthetic routes, each involving at least one different enzyme. Figure 3 organizes all known carboxylic Chl biosynthetic reactions into a logical scheme made up of 15 different biosynthetic routes.



    FourteenRoutesCarboxylicPW.jpg - 86601 Bytes


    Fig. 3. Integrated Chl a/b biosynthetic pathway depicting 15 carboxylic routes. To facilitate understanding of the text, various biosynthetic routes are designated by the numbers 1-15. Reproduced from Rebeiz et al, 2002.

    2. Thylakoid Apoprotein Biosynthesis

    The biosynthesis of thylakoid apoproteins is a very complex phenomenon. Some apoproteins are coded for by nuclear DNA, are translated on cytoplasmic ribosomes and are transported to developing chloroplasts. Other apoproteins are coded for by plastid DNA and are translated on chloroplast ribosomes. A detailed discussion of chloroplast apoprotein biosynthesis is beyond the scope of this discussion. The reader is referred to reference (Sundqvist et al, 1993) for a comprehensive discussion of this topic. For the purpose ofthis discussion it suffices to say that a PSU is an extremely complex structure that consists of many highly folded thylakoid and soluble proteins as well as membrane-bound pigment protein complexes having different functions in the light and dark steps of photosynthesis. An early visualization of a linear model of a PSU in the unfolded state is depicted in Fig. 4.

    PSU111006a.jpg - 158411 Bytes

    Fig. 4 . Schematics of a linear model of a PSU in the unfolded state Reproduced from reference (Bassi et al, 1990).

    3. assembly of chl-protein complexes

    Success in the bioengineering of smaller PSUs resides in a thorough understanding of how the Chl and thylakoid apoprotein biosynthetic pathways are coordinated to generate a specific functional Chl-protein complex. It is known for example that an apoprotein formed in the cytoplasm or in the chloroplast has to bind Chl molecules, has to fold properly, and has to wind up in the right place on the thylakoid in order to become a functional Chl-protein complex having a specific role in photosynthesis. What is unknown however is how an apoprotein formed in the cytoplasm or in the chloroplast becomes associated with Chl to become a specific Chl-protein complex of PSI, PSII or a light harvesting Chl-protein complex, having a specific function in photosynthesis. We have recently examined three possible models for that scenario, which have been referred to as: (a) the single-branched Chl biosynthetic pathway (SBP)-single location model, (b) the SBP-multilocation model, and (c) the multi-branched Chl biosynthetic pathway (MBP)-sublocation model. The models take into account the dimension of the PSU (Bassi et al, 1990), the biochemical heterogeneity of the Chl biosynthetic pathway (Rebeiz et al, 1994, 2002a), and the biosynthetic and structural complexity of thylakoid membranes (Sundqvist and Ryberg, 1993). The three models are described below.

    a. Assembly of Chl-Protein Complexes: the SBP-Single Location Model

    The SBP-single location model is depicted schematically below, in Fig. 5. Within the PSU, this model accommodates only one Chl-apoprotein biosynthesis center and no Chl-apoprotein biosynthesis subcenters. Within the Chl-apoprotein biosynthesis center, Chl a and b are formed via a single-branched Chl

    SBPSLM.jpg - 23426 Bytes
    Fig. 5. Schematics of the SBP-single location model in a PSU. As an example, the functionality of the model was illustrated with the use of three apoproteins namely CP29, LCHI-730 and CP47. Abbreviations: SBP = single-branched Chl biosynthetic pathway; PSII = photosystem II; LHCII, the major light-harvesting Chl-protein complex of PSII, LHCI, one of the LHC antennae of PSI, CP47 and CP29, two PSII antennae, LHCI-730, the LHC antenna of PSI. Curved lines indicate putative energy transfer between tetrapyrroles and a Chl-protein complex. Adapted from Kopetz et al, 2004.

    biosynthetic pathway (Fig. 2) at a location accessible to all Chl-binding apoproteins. The latter will have to access that locationin the unfolded state, pick up a complement of MV Chl a and/or MV Chl b, and undergo appropriate folding. Then the folded Chl-apoprotein complex has to move from the central location to a specific PSI, PSII, or Chl a/b LHC-protein location within the Chl-apoprotein biosynthesis center over distances of up to 225 (Kolossov et al, 2003, Kopetz et al, 2004). In this model, it is unlikely to observe resonance energy transfer between metabolic tetrapyrroles and some of the Chl-apoprotein complexes located at distances longer than 100 . This is because resonance excitation energy transfer takes place only over distances shorter than 100 (Calvert and Pitts, 1967).

    b. Assembly of Chl-Protein Complexes: the SBP-Multilocation Location Model.

    The SBP-Multilocation model is depicted schematically below, in Fig. 6. In this model, every biosynthetic location within the PSU is considered to be a Chl-apoprotein thylakoid biosynthesis center. In every Chl-apoprotein biosynthesis location, a complete single-branched Chl a/b biosynthetic pathway (Fig. 2) is active. Association of Chl a and/or Chl b with specific PSI, PSII, or LHC apoproteins at any location is random. In every Chl-apoprotein biosynthesis center, distances separating metabolic tetrapyrroles from the Chl-protein complexes are shorter than in the SBP-single-location model. Because of the shorter distances separating the accumulated tetrapyrroles from Chl-protein complexes, resonance excitation energy transfer between various tetrapyrroles and Chl-apoprotein complexes within each center may be observed. However, formation of MV Mg-Proto (Mp) and its MV methyl ester (Mpe) [i. e. Mp(e)] is not observed in any pigment-protein complex. This is because the single-branched Chl biosynthetic pathway does not account for MV Mp(e) biosynthesis.

    SBPMLM1.jpg - 66318 Bytes
    Fig. 6. Schematics of the SBP-Multilocation model in a PSU. All abbreviations and conventions are as in Fig. 5. Adapted from Kolossov et al, 2003 and Kopetz et al, 2004.

    c. Assembly of Chl-Protein Complexes: the MBP-Sublocation Model

    The SBP-sublocation model is depicted schematically below, in Fig. 7. In this model, the unified multibranched Chl a/b biosynthetic pathway, Rebeiz et al, 2003)is visualized as the template of a Chl-protein biosynthesis center where the assembly of PSI, PSII and LHC takes place. The multiple Chl biosynthetic routes are visualized, individually or in groups of one or several adjacent routes, as Chl-apoprotein biosynthesis subcenters earmarked for the coordinated assembly of individual Chl-apoprotein complexes. Apoproteins destined to some of the subcenters may possess specific polypeptide signals for specific Chl biosynthetic enzymes peculiar to that subcenter, such as 4-vinyl reductases, formyl synthetases or Chl a and Chl b synthetases. Once an apoprotein formed in the cytoplasm or in the plastid reaches its subcenter destination and its signal is split off, it binds nascent Chl formed via one or more biosynthetic routes, as well as carotenoids. During pigment binding, the apoprotein folds properly and acts at that location, while folding or after folding, as a template for the assembly of other pigment-proteins.
    MBPSubLM.jpg - 96852 Bytes
    Fig. 7 Schematics of the MBP-sublocation model in a PSU. All abbreviations and conventions are as in Fig. 5. Adapted from Kolossov et al, 2003 and Kopetz et al, 2004 Because of the shorter distances separating the accumulated tetrapyrroles from Chl-protein complexes, within each subcenter, resonance excitation energy transfer between various metabolic tetrapyrroles and Chl is readily observed. In this model, both MV and DV Mp(e) may be present in some pigment-protein complexes, in particular if more than one Chl biosynthetic route is involved in the Chl formation of a particular Chl-protein complex.

    4. Which Chl-Thylakoid Apoprotein Assembly Model is Favored by Experimental Evidence?

    In order to determine which Chl-thylakoid apoprotein assembly model is likely to be functional during thylakoid membrane formation, we tested the ompatibility of the three aforementioned models by resonance excitation energy transfer between anabolic tetrapyrrole intermediates of the Chl biosynthetic pathway and various thylakoid Chl-protein complexes. Fluorescence resonance excitation energy transfer involves the transfer of excitation energy from an excited donor D* to an unexcited acceptor A (Calvert and Pitts, 1967; Lakowicz, 1999; Turro, 1965). The transfer is the result of dipole-dipole interaction between donor and acceptor and does not involve the exchange of a photon. The rate of energy transfer depends upon (a) the extent of overlap of the emission spectrum of the donor and the absorption spectrum of the acceptor, (b) the relative orientation of the donor and acceptor transition dipoles, and (c) the distance between donor and acceptor molecules. The probability of resonance excitation energy transfer between donors and acceptors increases with time but is inversely proportional to the sixth power of the fixed distance separating the centers of the donor and acceptor molecules. It has been estimated that dipole-dipole energy transfer between donor and acceptor molecules may occur up to separation distances of 50 to 100 (Calvert and Pitts, 1967).

    Resonance excitation energy transfer from three tetrapyrrole donors to the Chl a of various Chl-protein complexes were monitored, namely: from Proto, Mp(e) and MV and DV Pchlide a. DV Proto is a common precursor of heme and Chl. It is the immediate precursor of DV Mg-Proto. As such, it is an early intermediate along the Chl biosynthetic chain. Biosynthetically, it is several steps removed from the Chl end product (Rebeiz et al, 2003). Mg-Proto is a mixed MV-DV, dicarboxylic tetrapyrrole pool, consisting of DV and MV Mg-Proto (Rebeiz et al, 2003). It is the precursor of DV and MV Pchlide a. The [(Pchl(ide)] of higher plants consists of about 95% Pchlide a and about 5% Pchlide a ester . The latter is esterified with long chain fatty alcohols at position 7 of the macrocycle. While Pchlide a ester consists mainly of MV Pchlide a ester, Pchlide a consists of DV and MV Pchlide a. The latter are the immediate precursors of DV and MV Chlide a(Rebeiz et al, 2003).

    Accumulation of the various tetrapyrrole donors was induced by incubation of green tissues with d-aminolevulinic acid (ALA) and/or 2,2-dipyridyl (Rebeiz et al, 1988). The task of selecting appropriate Chl a-protein acceptors was facilitated by the fluorescence properties of green plastids. At 77 K, emission spectra of isolated chloroplasts exhibit maxima at 683-686 nm (~F685), 693-696 nm (~F695), and 735-740 nm (~F735). It is believed that the fluorescence emitted at ~F685 nm arises from the Chl a of LHCII, the major thylakoid LHC antenna, and LHCI-680, one of the LHC antennae of PSI (Bassi et al, 1990). That emitted at ~F695 nm is believed to originate mainly from the Chl a of CP47 and CP29, two PSII antennae (Bassi et al, 1990). That emitted at ~F735 nm is believed to originate primarily from the Chl a of LHCI-730, a PSI antenna (Bassi et al, 1990). Since these emission maxima are readily observed in the fluorescence emission spectra of green tissues and are associated with definite thylakoid Chl a-protein complexes, it was conjectured that they would constitute a meaningful resource for monitoring excitation resonance energy transfer between anabolic tetrapyrroles and representative Chl a-protein complexes.

    To monitor the possible occurrence of resonance excitation energy transfer between the accumulated anabolic tetrapyrroles and Chl a-protein complexes, excitation spectra were recorded at 77K at the respective emission maxima of the selected Chl a acceptors, namely at ~685, ~695, and ~735nm. It was conjectured that if resonance excitation energy transfers were to be observed between the tetrapyrrole donors and the selected Chl a acceptors, definite excitation maxima would be observed. These excitation maxima would correspond to absorbance maxima of the various tetrapyrrole donors, and would correspond to the peaks of the resonance excitation energy transfer bands.

    <

    a. The SBP-single location model is not compatible with resonance excitation energy transfer between anabolic tetrapyrrole donors and chl a-proteins acceptors in chloroplasts

    .

    The compatibility of the SBP-single location model with the formation of Chl a-thylakoid proteins was tested by monitoring resonance excitation energy transfer between anabolic tetrapyrrole intermediates of the Chl biosynthetic pathway and various thylakoid Chl a-protein complexes. Pronounced resonance excitation energy transfer bands from Proto, Mp(e), and Pchl(ide) a to Chl a ~F685, ~F695, and ~F735 were detected (Table 1).

    Assignment of in situ resonance excitation energy transfer maxima to various metabolic tetrapyrroles was unambiguous except for a few cases at the short wavelength and long wavelength extremes of excitation bands. Contrary to previous beliefs, it was surprising to observe a significant diversity in the various intra-membrane environments of Proto, Mp(e), and Pchl(ide) a (Kolossov et al,2003 . This diversity was manifested by a differential donation of resonance excitation energy transfer from multiple Proto, Mp(e) and Pchl(ide) a sites to the different Chl a-apoprotein complexes. This in turn, is strongly compatible with the biosynthetic heterogeneity of the Chl biosynthetic pathway.

    Since resonance excitation energy transfer is insignificant at distances larger than 100 (Calvert and Pitts, 1967), the detection of pronounced resonance excitation energy transfer from Proto, Mp(e), and Pchl(ide) a to Chl a ~F685, ~F695, and ~F735 (Table 1), indicated that these anabolic tetrapyrroles were within distances of 100 or less of the Chl a acceptors. This in turn was incompatible with the functionality of the SBP-single location Chl-thylakoid biogenesis model as detailed below.

    The early concept of a PSU consisting of about 500 antenna Chl per reaction center has evolved into two pigment systems each with its own reaction center and antenna Chl (Allen and Forsberg, 2001; Anderson, 2002; Staehelin, 2003). The early visualization of the two photosystems consisted of various pigment-protein complexes arrayed into a linear PSU (the continuous array model), about 450 in length and 130 in width (Bassi et al, 1990 ). In the PSU, the LHCII was depicted as being shared between the two photosystems (Bassi et al, 1990). More recent models however, favor the concept of a laterally heterogeneous PSU (Allen and Forsberg, 2001; Anderson, 2002; Staehelin, 2003). In this model LHCII shuttles between PSI and PSII upon phosphorylation and dephosphorylation (Allen and Forsberg, 2001). Furthermore while PSII is mainly (but not exclusively) located in appressed thylakoid domains, PSI is located in non-appressed stroma thylakoids, grana margins, and end membranes (Anderson, 2002, Staehelin, 2003).

    The SBP-single location model is incompatible with the linear continuous array model, and the laterally heterogeneous PSU model. Indeed the SBP-single location model calls for Chl a and b to be formed via a single-branched Chl biosynthetic pathway at a location accessible to all Chl-binding apoproteins. The latter will have to access that location in the unfolded state, pick up a complement of MV Chl a and/or MV Chl b, and undergo appropriate folding. Then the folded Chl-apoprotein complex has to move from the central location to a specific PSI, PSII, or Chl a/b LHC-protein location within the Chl-apoprotein biosynthesis center over distances of up to 225 in the linear continuous array model, or over larger distances, in the laterally heterogeneous model, to become part of PSI, PSII or LHCII. If this were the case, then no resonance excitation energy transfer would be observed between anabolic tetrapyrroles and the various Chl-protein complexes, and the distances separating the anabolic tetrapyrroles from the various Chl-protein complexes would be much larger than the values reported in Table 1.

    b. incompatibility of the SBP-MLM model with experimental data

    The shorter distances separating anabolic tetrapyrroles from Chl-protein complexes reported in Table 1 are compatible with the SBP-multilocation and MBP-sublocation models. However, overwhelming experimental evidence argues against the operation of a single-branched Chl biosynthetic pathway in plants (Rebeiz et al, 2003).

    c. Compatibility of the MBP-SUBLM model with experimental data

    The above consideratiopns leaves the MBP-sublocation model (MBPSUBLM) as a viable working hypothesis. In this model (Rebeiz et al, 2003), the unified multibranched Chl a/b biosynthetic pathway, is visualized as the template of a Chl-protein biosynthesis center where the assembly of PSI, PSII and LHC takes place (Rebeiz et al, 1999, 2004). The multiple Chl biosynthetic routes are visualized, individually or in groups of one or several adjacent routes, as Chl-apoprotein biosynthesis subcenters earmarked for the coordinated assembly of individual Chl-apoprotein complexes. Apoproteins destined to some of the subcenters may possess specific polypeptide signals for specific Chl biosynthetic enzymes peculiar to that subcenter, such as 4-vinyl reductases, formyl synthetases or Chl a and Chl b synthetases. Once an apoprotein formed in the cytoplasm or in the plastid reaches its subcenter destination and its signal is split off, it binds nascent Chl formed via one or more biosynthetic routes, as well as carotenoids. During pigment binding, the apoprotein folds properly and acts at that location, while folding or after folding, as a template for the assembly of other pigment-proteins. This model is compatible with the lateral heterogeneity of the PSU and can account for the observed resonance excitation energy transfers (Table 1) and the short distances separating anabolic tetrapyrroles from Chl-protein complexes in the distinct PSI, PSII and shuttling LHCII entities that compose the PSU (Table 2).

    F. Epilogue

    Future research dealing with the bioengineering of smaller PSU sizes will have to use as a working hypothesis the MBP-sublocation Chl a-thylakoid protein biosynthesis model. The first order of business will have to deal with the determination of which Chl biosynthetic routes gives rise to PSI, PSII and LHCII Chl-protein complexes. The greening process may then be manipulated to bioengineer genetically modified plants with a smaller PSU, i. e. with more PSU units having less antenna Chl per unit thylakoid area. Nevertheless this type of agriculture using genetically modified plants with smaller PSU sizes and higher photosynthetic conversion efficiencies will still be at the mercy of extrinsic factors and weather uncertainties.

    In our opinion the ultimate agriculture of the future should consist of bioreactors populated with bioengineered, highly efficient photosynthetic membranes, with a small PSU size and operating at efficiencies that approach the 12% maximal theoretical efficiency of the PETS that may be observed under white light, or the 27% maximal theoretical efficiency that may be achieved under red light. Such conditions may be set up during space travel, in large space stations, or in human colonies established on the moon or on Mars(Rebeiz et al, 1982). The photosynthetic product may well be a short chain carbohydrate such as glycerol that can be converted into food fiber and energy.

    In the meanwhile, let us not forget that a journey of 10,000 miles starts with the first step.

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