What substance is used to temporarily store high energy electrons that were harvested from glucose molecules in the cytoplasm of the cell?

Light-Dependent Reactions [Z Scheme] [Neațu et al., 2014]

Light-dependent reactions happen in the thylakoid membrane of the chloroplasts and occur in the presence of sunlight. The sunlight is converted to chemical energy during these reactions.

The chlorophyll in the plants absorb sunlight and transfers to the photosystem which are responsible for photosynthesis.

Water is used to provide hydrogen ions and electrons but also produces oxygen.

The electrons and protons are used to produce NADPH [the reduced form of nicotine adenine dinucleotide phosphoric acid] and ATP [adenosine triphosphate].

ATP and NADPH are energy storage and electron carrier/donor molecule. Both ATP and NADPH are used in the next stage of photosynthesis.

The chlorophyll molecule regains the lost electron from a water molecule through a process called photolysis, which releases dioxygen [O2] molecule.

The light-dependent reactions can be expressed as:

2H2O+2NADP++3ADP+ 3Pi→O2+2NADPH+3ATP

4.2.1.1.3 The Photosynthetic Z-Scheme

To understand the light-dependent reactions of photosynthesis, it is best to start with reactions in PSII. Light energy is trapped by PSII causing an electron from P680 to be promoted to a higher energy level [an excited state]. This excited electron is rapidly transferred to a primary electron-acceptor molecule that is closely associated with P680. If this transfer does not occur immediately, the excited electron falls back to its ground state in P680, giving-off energy [fluorescence] in the process. From the primary electron acceptor, the electron is transferred from one acceptor to another within PSII. In this electron transfer chain, the energy of the excited electron is utilized to move protons [H+] from the stroma to the lumen of the thylakoids. Finally, a mobile electron acceptor carries the electron to PSI where it is transferred to P700. The photochemical reactions can be illustrated in the Photosynthetic Z-scheme [Fig. 4.5].

As electrons move on to Photosystem I, the pigment P680 [in Photosystem II] is depleted of electrons and becomes a powerful oxidizing agent capable of stripping electrons from water, thereby splitting the water molecule as follows:

2H2O→4H ++4e–+O2

Protons released from this reaction accumulate in the lumen of the thylakoids.

P700 [in PSI] also absorbs light energy and in so doing one electron [supplied by PSII] is promoted to the excited state. Again this excited electron is immediately transferred to a primary electron acceptor closely associated with PSI. From the primary electron acceptor, the excited electron moves across an electron transfer chain and is finally transferred to NADP+ resulting in the formation of NADPH as follows:

NADP ++2e–+H+→NADPH

This reaction occurs on the stroma side of the thylakoid membrane. NADPH is a powerful reducing agent, which means that it has a strong ability to force its electrons and hydrogen on to other molecules.

Introduction

During photosynthesis light energy is used to split water, generating O2 and electrons that are then used to produce the ATP and NADPH required for carbon fixation. Photosystem II [PSII] functions to capture light energy and transfer it to plastoquinone, the first molecule in an electron transport chain that leads to the production of ATP. The oxidized reaction center pigment P680 returns to a reduced state by stripping electrons from water in a process known as photolysis, which ultimately results in the production of O2. Photosystem I [PSI] is also capable of absorbing light energy. Electrons from its reaction center pigment P700 are transferred to the protein ferredoxin, which can then donate the electrons to the electron carrier NADP+ to form NADPH or to the electron transport chain resulting in the production of additional ATP.

ATP and NADPH produced by the light-dependent reactions of the photosystems are used by the Calvin cycle in the stroma of the chloroplast. Molecules of CO2 gas are fixed into molecules of 3-phosphoglycerate in a reaction catalyzed by the enzyme Rubisco. Subsequent reactions convert molecules of 3-phosphoglycerate into molecules of glyceraldehyde-3-phosphate, some of which will ultimately be converted into glucose in the cytoplasm of the plant cell.

Where in a eukaryotic cell do photosynthesis and the Calvin cycle occur?

Research/review the reactions associated with noncyclic photosynthesis. Identify where O2 and NADPH production occurs.

Research/review the reactions of the Calvin cycle. Identify where CO2 is consumed and glyceraldehyde-3-phosphate is produced.

Describe how a plant cell would use a molecule of glucose.

2.2 Adaptive strategies. Oxygen consumption—The Mehler reaction

In cyanobacteria, oxygen is potentially consumed through aerobic respiration and two light-dependent reactions, the oxygenase activity of RubisCO [photorespiration] and the photosynthetic reduction of O2, termed pseudocyclic photophosphorylation or the Mehler reaction [Box 35.2]. In contrast to terrestrial C3 plants, which have relatively high rates of photorespiration, photorespiration of oceanic phytoplankton is usually low when dissolved inorganic carbon concentrations in seawater are at equilibrium [∼2 mM] with the atmosphere. Moreover, cyanobacteria operate a CO2 concentrating mechanism [CCM] which raises the CO2 concentration in the vicinity of RubisCO and inhibits oxygenase activity [Kaplan and Reinhold, 1999].

Box 35.2

The Mehler reaction [pseudocyclic photophosphorylation]

In 1951, the late Alan Mehler [1951] observed that chloroplasts can use oxygen as an electron acceptor. The reaction sequence is

H2O+2O2→2O2 +2H++12O2,

2O2+2H+→H2O2+O2

and, in the presence of catalase:

H2O2→H2O+12O2.

Net gas exchange is absent since the overall electron transport reaction which involves both Photosystems II and I is:

H2O+2O2→H2 O+2O2

The Mehler reaction is a photochemical reduction of O2 to H2O2 or H2O in photosystem I [Box 35.2]. Mehler activity is thought to be a mechanism for energy dissipation under high light intensities or when carbon fixation is limited by supply of inorganic carbon [Helman et al., 2003]. Since the products of O2 reduction are often superoxide and hydrogen peroxide [with superoxide dismutase catalyzing the reduction of superoxide to peroxide], Mehler activity has been hypothesized to be a metabolic defect rather than an adaptive strategy [Patterson and Myers, 1973]. However, in the cyanobacterium Synechocystis sp. [PCC 6803], superoxide is reduced directly to water without a hydrogen peroxide intermediate [Helman et al., 2003]. This single step reduction of superoxide to water is catalyzed by A-type flavoproteins; two of which [flv1, flv3] were identified as essential for this activity [Helman et al., 2003]. Examination of the genome of Trichodesmium, identifies homologous genes to flv1 and flv3 with 62% and 67% sequence identity, respectively [Milligan et al., 2007].

Mehler activity is generally considered a process which can only consume photosynthetically derived O2, and it cannot cause net consumption of O2 because PSI activity relies on photosynthetically derived electrons [Kana, 1993]. Yet, the shared-arrangement of photosynthetic and respiratory electron transport chains in cyanobacteria allows electrons from respiratory derived NAD[P]H to feed into the plastoquinone pool of the photosynthetic electron transport chain and reduce PSI [Schmetterer, 1994]. Through the translocation of reductant [i.e. glucose 6-phosphate] from cells with functional PSII, Mehler activity can result in a net consumption of O2 in cells [or heterocysts] which have no PSII activity and in which nitrogen is fixed [Fig. 35.3].

Results from Trichodesmium provide an example of the Mehler pathway's role in modulating oxygen and facilitating nitrogen-fixation. In this species, under nitrogen-fixation conditions, approximately 50% of gross O2 production is consumed through Mehler activity [Fig. 35.4]; this is about twice the rate reported for Synechococcus [∼25% of gross O2 production] exposed to photoinhibitory irradiances [Kana, 1992]. Mehler activity is dependent both on the time of day and the nitrogen source [Fig. 35.4]. The period of maximum N2 fixation is coincident with a decline in the net production of O2 and a rise in the consumption of oxygen via Mehler activity, which is consistent with the hypothesized role of this pathway as a mechanism to protect nitrogenase from O2 damage. In nitrate-grown Trichodesmium cultures [with negligible nitrogen-fixation], Mehler activity increases with light induction, but quickly drops to low and constant rates [10% of gross production] through the rest of the photoperiod [Fig. 35.4].

In Trichodesmium, Mehler activity is essential, as Trichodesmium relies on short term regulation of PSII and nitrogenase activities to separate these functions within a trichome [Berman-Frank et al., 2003]. PSII activity is regulated on time scales of 10–15 min and appears to involve the association/disassociation of the light harvesting complex [LHC II] from PSII [Küpper et al., 2004]. Nitrogenase activity is also regulated on similar time scales when incubated at different oxygen concentrations, with transcriptional and translational regulation requiring longer time-scales and higher concentrations of exposure [Fig. 35.2]. While PSII activity is repressed in N2 fixing cells, the activity of PSI is responsible for net O2 consumption relying on the translocation of reductant for the donor side of PSI and the flux of photons driving the oxidation. Cultures of Trichodesmium grown under low [5%] oxygen showed some nitrogenase activity during the night; this activity was absent in controls [21%] and in high [50%] oxygen cultures [Küpper et al., 2004]. The lack of Mehler activity at night in the controls [21%] and in the high oxygen cultures may thus reduce the total possible oxygen consumption and prevent nitrogenase activity. At lower O2 concentrations, N2 fixation can proceed in darkness because the respiratory rates are sufficient to consume O2.

1.2.1 Photosynthesis

In photosynthesis a blue/green substance called chlorophyll A and a yellow/green substance called chlorophyll B use light energy [normally sunlight but sometimes artificial] to change carbon dioxide and water into sugars [carbohydrates] and oxygen in the green parts of the plant. The amount of photosynthesis per day which takes place is limited by the duration and intensity of sunlight, and the ability of the green parts of a plant to capture it. The amount of carbon dioxide available can also be a limiting factor. Shortage of water, low temperatures and leaf disease or damage can reduce photosynthesis, as can shading by other plants, e.g. by weeds in a crop. The cells that contain chlorophyll also have orange/yellow pigments such as xanthophyll and carotene, and brown pigments called phaeophytins which absorb different wavelengths of light than the chlorophylls. Crop plants can only build up chlorophyll A and B in the light, and so any leaves that develop in the dark are yellow and cannot efficiently produce carbohydrates. The yellowing of leaves [chlorosis] can also be caused by disease attack, nutrient deficiency or natural senescence [dying off].

Oxygen is released back into the atmosphere during photosynthesis and the process may be set out as follows:

The light reaction [light dependent]

This takes place in the thylakoid membranes inside the ‘chloroplast’, an organelle found inside the cells of green tissue. Light provides energy for the chlorophyll molecule that releases electrons. These split water into oxygen and hydrogen.

The chemical reaction of this stage is:

[1.1]2H2O→ 2H2+O2

The hydrogen then moves into the next stage:

The dark reaction [light independent]

This takes place in the watery stroma of the chloroplast. Here the hydrogen is combined with carbon dioxide by the Calvin Cycle to give carbohydrate and water:

[1.2]2H2+CO2→CH2O+H2O

The carbohydrates are simple sugars, which can be moved through the vascular system of the plant in solution to wherever they are needed. This process not only provides the basis for all food production but it also supplies the oxygen which animals and plants need for respiration. The simple carbohydrates, such as glucose, may be built up to form starch for storage purposes or as cellulose for building cell walls. Fats and oils [lipids] are formed from carbohydrates by a process of esterification which produces mostly triglycerides. These are usually found in seeds and are a form of concentrated energy. Protein material, which is an essential part of all living cells, is made from carbohydrates and nitrogen compounds and also frequently contains sulphur. These form amino acids which are held together in proteins by peptide bonds.

Most plants consist of roots, stems, leaves and reproductive parts and need a medium in which to grow. These media could be soil, compost, water where plants are grown hydroponically or even air, where the bare roots are sprayed with a fine mist of nutrients and water [aeroponics]. In soil the roots spread through the spaces between the particles and anchor the plant. The amount of root growth can be phenomenal. For example, in a single plant of wheat the root system may extend to many miles.

The leaves, with their broad surfaces, are the main parts of the plant where photosynthesis occurs [Fig. 1.1]. A very important feature of the leaf structure is the presence of large numbers of tiny pores [stomata] on the surface of the leaf [Fig. 1.2]. There are usually thousands of stomata per square centimetre of leaf surface. Each pore [stoma] is oval-shaped and surrounded by two guard cells. The carbon dioxide used in photosynthesis diffuses into the leaf through the stomata. Most of the water vapour leaving the plant, as well as the oxygen from photosynthesis, diffuses out through the stomata.

2.1 Synthesis and Degradation

The hormone precursor, vitamin D3, can be obtained from the diet or synthesized from 7-dehydrocholesterol in skin in a UV light-dependent reaction [Fig. 1]. Vitamin D3 then circulates to the liver, where it is converted to 25-hydroxyvitamin D3 [25D], the major circulating form that is assayed to quantitate clinical vitamin D status. The final step in the production of the hormonal form occurs mainly, but not exclusively, in the kidney, via a tightly regulated 1α-hydroxylation reaction catalyzed by mitochondrial CYP27B1 [Fig. 1]. The major inducer of CYP27B1 in kidney is parathyroid hormone [PTH] that is secreted during hypocalcemia [Hughes, Brumbaugh, Haussler, Wergedal, & Baylink, 1975]. When 1,25D levels then rise, PTH synthesis in the parathyroid glands is suppressed by a direct action of 1,25D-liganded VDR on gene transcription [DeMay, Kiernan, DeLuca, & Kronenberg, 1992]. This negative feedback loop [not shown in Fig. 1] is vital to curtail the bone-resorbing effects of PTH in anticipation of 1,25D-mediated increases in both intestinal calcium absorption and bone resorption, thus preventing hypercalcemia. The major repressor of CYP27B1 in kidney is FGF23, the phosphaturic peptide hormone secreted during hyperphosphatemia [Bergwitz & Juppner, 2010]. We [Kolek et al., 2005] and others [Quarles, 2008] proved that 1,25D induces FGF23 release from bone osteocytes in a process that is independently stimulated by high circulating phosphate levels. Thus, PTH is repressed by 1,25D and calcium, whereas FGF23 is induced by 1,25D and phosphate, protecting against hypercalcemia and hyperphosphatemia, respectively, either of which can elicit ectopic calcification. Finally, a second inducer of CYP27B1 is phosphate depletion [Hughes et al., 1975], a phenomenon we now understand to be mediated by relief of FGF23-mediated suppression of CYP27B1, since FGF23 is no longer secreted under low phosphate conditions.

Also illustrated in Fig. 1 [center right] is the mechanism that initiates the process of 1,25D catabolism in all target cells, namely the action of CYP24A1 [St-Arnaud, 2010]. The CYP24A1 gene is transcriptionally activated by 1,25D [Ohyama et al., 1994; Zierold, Darwish, & DeLuca, 1994], as well as by FGF23 [Shimada, Hasegawa, et al., 2004]. Thus, the vitamin D endocrine system is elegantly choreographed by feedback controls that interpret bone mineral ion status to prevent bone mineral excess as well as hypervitaminosis D. The vitamin D intracrine system, in contrast, appears to be more dependent on the availability of ample 25D substrate to generate 1,25D locally in order to maintain healthy epithelial, immune, cardiovascular, and nervous systems.

A Case History: Mammalian PRE.

A mammalian photoreactivating enzyme was first reported in human leukocytes: the activity was shown to cause the disappearance of dimers in DNA in a light-dependent reaction, to be trypsin-sensitive, and to have an apparent molecular weight of about 40,000 [14]. Additional studies showed that the enzyme had an action spectrum extending from 300 nm to at least 577 nm, with maximum at about 400 nm, and that its action converted dimers to pyrimidine monomers [15]. Photoreactivating enzyme activities have been found not only in human leukocytes, but also in bovine bone marrow, in human and murine cells in culture [5], as well as in canine, feline, and bovine corneal cells [16]. If this enzyme is present in the cell, can it act on dimers in cellular DNA? For the case of human fibroblasts cells in culture, several studies have shown that the cells can monomerize pyrimidine dimers in their DNA [17, 11, 12, 15]. Action spectra for dimer photoreactivation in human fibroblasts have also been determined: the spectra extend from about 300 nm to at least 577 nm, with a maximum about 400 nm. These spectra agree well with those for dimer monomerization by the human leukocyte PRE in vitro, indicating that the human PR enzyme mediates cellular dimer photoreactivation [18].

Does the action of this PRE mediate biological recovery? This point is particularly important in view of reports that photosensitizers which can produce dimer reversal not only do not mediate biological photoreactivation but instead cause additional inactivation, presumably through formation of new photoproducts [19]. The work of Dr. Helga Harm first indicated that mammalian PREs could restore biological activity to transforming DNA [20]. Wagner et al. showed that plaque-forming ability of uv-irradiated herpes simplex virus could be restored by photoreactivation in cultured human fibroblasts [21]. Sutherland and Oliver have also reported photoreactivation of DNA synthesis inhibition in human fibroblasts [22].

For the case of human cells, one further question is important: does photoreactivation function in DNA repair in man? It has been suggested that the dimer monomerizing activity observed in cultured human cells might result from a component of the culture medium taken up by the cells. Several lines of evidence argue against such a possibility: First, neither medium nor serum components show any photoreactivating activity [15]. Second, photoreactivating enzyme activity has been found in tissues taken directly from various mammals and never exposed to culture media [14, 15, 16, 20]. Third, action spectra for PR by human fibroblasts [which were grown in culture medium] agree closely with those for PR activity by the human leukocyte enzyme, [which was not exposed to culture medium] [18]. In addition, van der Leun and Stoop have presented evidence for photoreactivation of erythema in human skin [23]. We have thus looked for dimer photoreactivation in intact human leukocytes: immediately after withdrawal of the blood sample, erythrocytes are separated from leukocytes by sedimentation; the leukocytes are washed in phosphate-buffered saline, and exposed to 254 nm radiation, then kept in the dark or exposed to broad spectrum photoreactivating light. Samples are then analyzed by DNA extraction, M. luteus uv-endonuclease treatment, and electrophoresis in alkaline agarose gels. Figure 1 shows that photoreactivation decreases the number of uv-endonuclease-sensitive sites in cellular DNA.

Photosynthesis and starch synthesis

Chloroplasts use photosynthesis to manufacture low-molecular-weight, reduced carbon compounds, commonly called sugars. In brief, photosynthesis can be divided into two distinct sets of reactions [Hoober, 2006]. The ‘light-dependent reactions’ harvest light energy and use that energy to transport electrons through an electron transport chain embedded in the thylakoid membrane. Chlorophyll is the primary photosynthetic pigment; hence, thylakoid membranes are deep green in color. The light-dependent reactions synthesise ATP and the reductant NADPH. Subsequently, the ‘light-independent reactions’ use that NADPH and ATP to reduce and phosphorylate oxidized atmospheric carbon to the level of a sugar phosphate.

The ‘light-dependent reactions’ have an electron transport chain consisting of two photosystems, PSII and PSI, connected by a series of redox-active lipids, proteins, and metal cofactors [Figure 3]. Each photosystem has as its core a reaction center [RC] surrounded by antenna complexes containing 300–400 molecules of chlorophyll, several molecules of carotenoid, and numerous pigment-binding proteins. When a chlorophyll molecule absorbs the energy of a photon, that energy is transferred from chlorophyll to chlorophyll through the antenna complex until it is ultimately absorbed by the reaction centers of PSII [P680] or PSI [P700]. Note that the antenna does not contain a redox chain – electrons are not being transported, only the absorbed energy. The RC is made of two special chlorophyll molecules that are capable of performing a ‘photoact,’ i.e. undergoing oxidation and reducing the electron transport chain. The electron ejected from P680 of PSII ultimately reduces a molecule of the lipid plastoquinone [PQ]. When doubly reduced with two electrons and two protons, the resulting PQH2 debinds from PSII, diffuses to and reduces the cytochrome b6/f complex [Cyt b6/f]. The oxidized P680 becomes re-reduced by drawing an electron out of the adjacent water splitting apparatus [WSA]. When four electrons have been extracted by PSII [requiring the absorbed energy of four photons], four protons and one molecule of O2 are released by the WSA [Figure 3 is drawn based on a stoichiometry of two electrons per one NADPH]. The absorption of a photon by a PSI antenna chlorophyll drives a second photoact, ultimately reducing NADP+ to NAPDH. Oxidized PSI extracts an electron from plastocyanin [PC], which is re-reduced by electrons from the cytochrome b6/f complex. Thus, whole-chain photosynthetic electron transport uses the energy of sunlight to oxidize low energy water, and reduce high-energy NADPH.

ATP is also synthesized by the light-dependent reactions, via chemiosmosis. The transthylakoid pH gradient needed to drive chemiosmosis is generated by two mechanisms: [1] the oxidation of one molecule of water produces two free protons, and [2] proton pumping via the PQ pool. When electrons coming from PSII reduce plastoquinone, the two protons needed to complete the reaction come from the stromal side of the thylakoid membrane. Then, when the resultant PQH2 is oxidized, two electrons enter the cytochrome b6/f complex the two protons are delivered to the thylakoid lumen. In this way, photosynthetic electron transport generates protons in the lumen and pumps protons across the thylakoid membrane, lowering the lumenal pH from roughly 7 in the dark to approximately 5 in the light. This substantial pH gradient is used to synthesise ATP in the light.

The light-independent reactions are located in the stroma and use the ATP and NADPH produced by the thylakoid membrane to reduce and phosphorylate atmospheric CO2 to the level of glyceraldehyde-3-phosphate [G3P]. Photosynthesis does not make glucose as an end product. It makes G3P. The resulting triose phosphate can be exported from the chloroplast and be translocated to all parts of the plant. Alternatively, if photosynthesis is running faster than translocation, the G3P is temporarily stored within the chloroplast as starch. Starch levels are typically high at the end of the light period, and low at pre-dawn. ‘Photosynthesis’ literally means to use light to fuel an anabolic process. In that regard, photosynthesis makes carbohydrates, amino acids, proteins, lipids, and nucleic acids – every reduced-carbon molecule in the plant.

Photosynthesis

It is most likely that the higher vascular plants evolved approximately 400 million years ago via photosynthesizing bacteria, algae, mosses and lichen. Photosynthesis is the key reaction in the evolution of higher plants, e.g. the tea plant, Camellia sinensis L. [Theaceae], and plant-derived substances as tea flavanols. Photosynthesis [discovered by Joseph Priestley, 1770, and Jan Ingenhousz, 1779] is probably the most important chemical reaction taking place on Earth. In photosynthesis, light energy is converted into chemical energy, and this is considered to be the ultimate source of energy sustaining life on Earth. The process can be described as two subsequent chemical reactions, a light-dependent reaction and the Calvin cycle. The two are linked together and controlled by enzymes. The light-dependent reaction is a photochemical reaction taking place in the thylakoid membranes of chloroplasts, where light energy is transformed into adenosine triphosphate [ATP] and nicotinamide adenine dinucleotide phosphate [NADPH].

The Calvin cycle [discovered by Melvin Calvin] takes place in the stroma of the chloroplast, and here, energy in the form of ATP and NADPH from the light-dependent reaction is used to convert carbondioxide to carboxyhydrates, namely 2 glyceraldehyde-3-phosphate [Figure 6.1], in a biochemical reaction. In order for the Calvin cycle to continue, two-thirds of the 2 glyceraldehyde-3-phosphate molecule is regenerated, so creating one glucose molecule requires six turns of the Calvin cycle. In summary, glyceraldehyde-3-phosphate is synthesized by the light-dependent reaction, and the Calvin cycle is used to form carbohydrate substances, e.g. starch and cellulose, which are essential for the plant.

1 Introduction

Carbon fixation is the process by which plants and algae convert the carbon found in inorganic molecules in the atmosphere into organic matter to produce biological building blocks and fuel for cellular respiration. Whilst heterotrophs rely on breaking down existing organic materials via food and digestion, for both energy and growth, photoautotrophy allows plants to use the light energy captured in the light dependent reaction [see Chapter 2] to drive the fixation of carbon from carbon dioxide, with the help of the most abundant protein on Earth, the enzyme rubisco.

The light reactions convert energy from the sun into chemical energy in the form of ATP and reductant [NADPH] needed for carbon fixation, often referred to as the light independent reaction, Calvin–Benson–Bassham Cycle [CBBC] or the C3 cycle. The CBBC is an autocatalytic cycle regenerating the original acceptor molecules as part of the process and can be divided into three phases: carboxylation, reduction, and regeneration. In this chapter, we will describe each of these phases, as well as variation that exists in photosynthetic pathways. Carbon fixation in C3 plants is usually limited by the concentration of CO2 [[CO2]] at the carboxylation sites [CC] inside the chloroplasts [Evans et al., 2009]. To reach the site of carboxylation, CO2 must travel from the atmosphere to rubisco and there are many limitations to this diffusion pathway [Ouyang et al., 2017] that can greatly influence the photosynthetic rate, and we will start by describing the various resistance components along this route.