It should be noted that the active study of photorespiration was carried out in the period when the biochemical mechanism of carbon dioxide assimilation, the so-called Calvin-Benson cycle, was already known [8]. With the discovery of this cycle, the mechanism of the binding of carbon dioxide molecule and the regeneration (reproduction) of its acceptor ribulose bisphosphate (RuBP), which initially adds CO₂, became clear. There was little information about the fate of individual participants in this process outside the CO₂ recovery cycle. It has been shown [9] that during photosynthesis, sucrose is synthesized from CO₂, which is transported to organs (roots, fruits, growth points) consuming products of photosynthesis.

In sink organs, a variety of substances needed by these tissues is synthesized from sucrose carbon. It was also found that the enzyme catalyzing the reaction of the addition of carbon dioxide is the most wide-spread protein in nature. More than half of the soluble proteins in the plant leaves are represented by an enzyme that fixes carbon dioxide, ribulose-bisphosphate carboxylase (RuBP carboxylase).

Since the first product of the assimilation of CO₂ in most plant species is the three-carbon compound -3-phosphoglyceric acid (PGA), this type of photosynthesis was called the C-3 type. The other, C-4 type of photosynthesis will be described below. The resulting PGA is then reduced to three carbon sugars (triosophosphates), whose carbon, having passed a series of transformations through various phosphoric esters of sugars (PES), is used in part to regenerate the CO₂ acceptor molecule (RuBP) (highlighted in red in Figure 3), but mainly to form sucrose as the major transport compound in most plant species.

It should be remembered that photosynthesis itself consists of two stages (Figure 3): the light stage, which includes photophysical and photochemical reactions of absorption and storage of the photon energy, and the dark reaction of carbon dioxide assimilation. The light stage (highlighted in blue in Figure 3) results in the formation of a strong reducing agent ̶ reduced nicotinamide adenine dinucleotide phosphate (NADPH) and energy-rich adenosine triphosphate (ATP). Formed during photochemical light reactions, NADPH and ATP are also called the “assimilatory force” of chloroplasts, since these substances are used in the course of dark biochemical reactions for the assimilation of CO₂ and the formation of sugars. The building of an “assimilatory force” is associated with electron transfer in the electron transport chain (ETC) of chloroplasts, when a chlorophyll molecule excited by photons generates an electron flow from water (which decomposes with the formation of oxygen) to NADP (Figure 3) through the ETC. As a result, NADP is reduced, and ATP is formed from adenosine diphosphate (ADP) in the process of photophosphorylation coupled with the electron flow. ETC consists of a series of components, differing in their redox potentials.

As it turned out, the CO₂ binding enzyme has a dual function (Figure 4). It can carry out both the carboxylation reaction (carbon dioxide addition to the five-carbon compound ribulose bisphosphate), followed by the formation of a six-carbon compound, which breaks down into two molecules of the three-carbon PGA) and the oxygenation reaction (the addition of an oxygen molecule with the formation of only one PGA molecule and one two-carbon glycolic acid phosphate molecule). Accordingly, this enzyme is usually designated as RuBP carboxylase/oxygenase or Rubisco (ribulose bisphosphate carboxylase/oxygenase).

Special studies have shown [11] that initially the carbon dioxide acceptor RBP “sits down” at the active site of the Rubisco enzyme, and then either CO₂ or O₂ can be bound and either carboxylation or oxygenation is performed (Figure 4). As a result, there is a competition between oxygen and carbon dioxide for the active center of the enzyme. The ability of this enzyme to bind carbon dioxide is significantly higher than oxygen. Therefore, with natural atmospheric concentrations of these gases (21% oxygen, and 0.03% carbon dioxide), the latter has significant advantages and the proportion of photorespiration in the leaf photosynthetic gas exchange under these conditions is only 25% - 30%.

However, if the concentration of carbon dioxide decreases and that of oxygen increases, then the competitiveness of oxygen grows and the role of photorespiration in the leaf gas exchange increases. In an extreme situation (complete absence of carbon dioxide at a natural oxygen concentration), photorespiration exceeds photosynthesis, and during the measurement of the leaf gas exchange in the light, carbon dioxide is not absorbed but released (see Figure 2, curve 2). In fact, under natural conditions, both release and absorption of CO₂ and O₂ occur, since both processes occur simultaneously, but release of O₂ is more intense than its absorption.

Studies of the further fate of phosphoglycolate carried out with the help of labeled atoms showed [12] that after removal of the phosphate residue glycolic acid (glycolate) is transferred from chloroplasts to the cytoplasm, where it is first oxidized to glyoxylic acid in special cellular structures (peroxisomes and mitochondria), and then is converted into the amino acid glycine by amination (Figure 3). In the mitochondria, a three-carbon amino acid serine is formed from two glycine molecules; at the same time one molecule of carbon dioxide is released.

Reactions of the glycolate formation and its further transformation proceed in the dark. Therefore, after turning off the light, when the process of carbon dioxide absorption and the restoration of its primary fixation products ceases, the formation and metabolism of glycolate continue, and the carbon dioxide resulting from the conversion of glycine to serine can still be released from the photosynthetic object. This is the very same CO₂ burst that occurs after turning off the light. In the light, CO₂ released during photorespiration reduces the observed photosynthetic rate, and at low carbon dioxide concentrations are the cause of the CCP existence.

Serine, which is formed during glycolate metabolism, is either used in the synthesis of proteins, or, after passing a series of biochemical reactions and losing the amino group, is converted into phosphoglyceric acid, which, in the fully grown leaves in light is usually involved in the photosynthetic reduction process in chloroplasts to form sugars [10]. Thus, as it can be seen in the diagram (Figure 3), the reactions of glycolate formation and further conversion, associated with photorespiration, allow bypassing the photosynthetic carboxylation reaction in the Calvin cycle, and the cycle can function without CO₂ absorption. This was the basis for the emergence of the first and most widely recognized hypothesis explaining the physiological meaning of the photorespiration existence.

Much work has been done in this direction, and such forms have been obtained [14]. The reactions related to glycolate transformations were blocked in these mutants. As a result, such plants turned out to be non-viable under the usual oxygen concentration in ambient air and could exist only at low oxygen concentration (less than 2%).

Searching for substances that inhibit glycolate metabolism and photorespiration. This direction was developed very intensively, since such inhibitors would allow studying deeper the very processes of photosynthesis and photorespiration. Such substances have been found (for example, glycidic acid) [14]. Plants treated with this preparation, reacted less to a decrease in oxygen concentration. In some cases, they turned out to have higher photosynthesis, but their growth was suppressed and they had low productivity.

A long search has not resulted in detecting species with reduced photorespiration among C3 plants, but the fact that there exist plants with a different type of photosynthesis was established. It turned out that the vast majority of plant species assimilate carbon dioxide along the so-called C3 path of photosynthesis, but not all plant species. There is a group of plants assimilating carbon dioxide by another mechanism. In these plants, another carbon dioxide acceptor is used—the three-carbon compound phosphoenolpyruvate (PEP), and the primary product of carbon dioxide assimilation is the four-carbon compounds. The primary product of carbon dioxide fixation in these plants is oxaloacetic acid (OAA), but this compound is unstable and quickly turns into another four-carbon compound, usually either malic acid or aspartic amino acid. Accordingly, this type of photosynthesis was called the C-4 type (Figure 6).

There are more than a thousand of such plant species. Among them are many tropical cereals (including corn, sugar cane, sorghum), the amaranth family, and some plant species from other families. Plants were also found with an intermediate type of photosynthesis, in which C-3 photosynthesis is observed under certain conditions, and C-4 photosynthesis reveals itself under others (most often under unfavorable (drought) conditions). The discovery of the C-4 type of photosynthesis strongly activated investigations of photorespiration, because C-4 plants did not show pronounced photorespiration.

The specific feature is that chloroplasts containing chlorophyll in these plants are not only in the leaf mesophyll (the main photosynthetic tissue), but also in the so-called bundle sheath cells that surround the phloem vessels transporting photosynthetic products from the leaf to the consuming organs. In the mesophyll cells of these plants (Figure 6), CO₂ is assimilated with the formation of four-carbon compounds (carboxylation process), which are then transferred to the bundle sheath cells.

In bundle sheath cells, these compounds (malic or aspartic acid) undergo decarboxylation (the process of CO₂ release that is reverse to carboxylation). The released carbon dioxide is reabsorbed with the help of Rubisco, since the normal Calvin-Benson cycle functions in the bundle sheath cells. This shows that in plants with the C-4 type of photosynthesis, the processes of the CO₂ carbon binding and its reduction to sugars are separated in space. In the mesophyll cells,

CO₂ absorption takes place, and in the bundle sheath cells it is reduced to sugars, which pass into conducting vessels for export to the consuming organs.

CO₂ binding in the mesophyll occurs with the help of phosphoenolpyruvate carboxylase, which has a high affinity for CO₂. Therefore, it can absorb it even at very low concentrations. Since the product of CO₂ fixation (malic or aspartic acid) is transferred into the bundle sheath cells by active transport across the cell membranes, CO₂ seems to be pumped into the bundle sheath cells. Here, CO₂ is assimilated by the usual mechanism incident to C-3 type plants, but occurs already at an elevated concentration of CO₂, formed by decarboxylation of malic or aspartic acid. This explains the lack of visible photorespiration in C-4 plants, since near Rubisco the concentration of CO₂ is significantly higher than in the ambient air and oxygenase reaction is suppressed, although the formation and metabolism of glycolate in these plants exist.

Thus, none of the directions in the search for ways to reduce photorespiration did not result positively in increased plant productivity. Moreover, it appeared that the conditions suppressing photorespiration also reduce the productivity of plants. These efforts show that in any case, it is impossible to interfere with the evolutionarily established process without careful analysis of the purpose for which it was created by nature. Further sections of this article are devoted to this issue.

It should be noted that the understanding of the role of photorespiration in the plant productivity among plant biologists has not changed almost since the 80s of the twentieth century. New publications that have appeared. Rakhmankulova and others [2] [3] [15] are dedicated to particular issues and continue to manipulate photorespiration in order to force productivity by suppressing this process. All recent reviews on photorespiration ignored the publications in which transketolase pathway was shown as a way of glycolate formation (alternative to Rubisco) by ¹⁴C kinetic experiments [16] [17] [18]. In the case of the formation of glycolate through the transketolase mechanism, a superoxide radical is required, which is formed in connection with the flow of electrons in the electron transport chain of chloroplasts. It is this mechanism that integrated photorespiration into the general regulation of photosynthesis in the whole plant system.

Interest in the photorespiratory process has been enhanced by the studies of the relationship between photosynthetic carbon metabolism (PCM) and transport of labeled assimilates from the leaf. It was established [16] [17] [18] that under the action of any photosynthesis suppressing factor, the export of assimilates from the leaf decreases and the non-carbohydrate orientation of the PCM increases. This phenomenon is especially pronounced under the action of nitrate nitrogen [20], the removal of some of the sinks for assimilates, or when comparing leaves of different ages [16]. The degree of closure of the glycolate cycle on the Calvin cycle and the source of glycolate formation change [16] [10].

This was confirmed by kinetic experiments on the incorporation of ¹⁴C into glycolate. When studying the dynamics of incorporation of ¹⁴C labeled carbon into photosynthetic products in leaves of plants in different physiological states, for example, after removing of fruits or increasing the level of nitrate nutrition [16] [21], there is a difference in the kinetic curves of ¹⁴C incorporation into glycolate. At an early stage (a few seconds in ¹⁴CO₂), the kinetic curves for the label incorporation into glycolate [17] [18] in different variants run in opposite directions (Figure 7). In the case of the glycolate formation through Rubisco (steady state of control plants), the label content in glycolate increases up to 20 seconds and then decreases. With other sources of glycolate formation (at removal of assimilate acceptors or increase in CO₂ concentration), the maximum label incorporation into glycolate is noted in the very first seconds, and then the label content rapidly decreases.

Transketolase reaction in the Calvin cycle may be another (apart from RuBPo) possible glycolate source [22]. In this case, the oxidizer is not the oxygen, but a superoxide radical formed either in the Mehler reaction [22] or in the nitrite reduction [23]. Even when reducing the oxygen level in the atmosphere of the test leaf to 1% and increasing the carbon dioxide concentration (up to 0.3%), the of glycolate metabolism product formation increases in the plants fed with nitrates, and decreases twofold without nitrates [23].

For the formation of labelled ¹⁴C-glycolate, the superoxide radical should oxidise the sugar-phosphate formed from the newly labelled ¹⁴C of the PGA, and then reduced to ¹⁴C-sugar-phosphate. With the oxygenase reaction of glycolate formation, the sugar-phosphate formed from the previously labelled ¹⁴C of the PGA should undergo 13 reduction and conversion reactions to obtain the RuBP, which will give the labelled ¹⁴C-glycolate. Therefore, in the RuBPo reaction of glycolate formation, the glycolate is labelled with a delay (Figure 7), because, first of all, all compound pools of the Calvin Cycle should be saturated with the labelled carbon.

Glycolate formation is a regulatory mechanism of photosynthetic function control in case of imbalance between light and dark reactions in chloroplasts. Such mechanism is likely to work in the sheath cells of C-4 type of plants. The researchers did not pay ample attention to this alternative glycolate source and, accordingly, to another aspect of participation of photorespiration in the photosynthesis regulation, despite the long-discovered transketolase mechanism of glycolate formation [22].

Our studies of the photosynthetic carbon metabolism in connection with the study of photorespiration and the nature of the Warburg effect have shown [23] [10] that the maximum carbon filling of the glycolate cycle (in ng/cm²∙s) occurs at simultaneously high illumination, concentration of CO₂ and oxygen [23]. In these circumstances, the delivery of ¹⁴C in the glycolate metabolism products is twice as much as at ambient CO₂ concentrations [23]. This is probably due to the known [24] electron transport stimulation in chloroplasts with increasing CO₂ concentration. It also shows that the intensity of carbon flow through the glycolate pathway is determined not only by the oxygen and the RuBPo activity, but also by the presence of superoxide radical, which is involved in the transketolase mechanism of glycolate formation. The oxygen endogenously formed in the chloroplasts or the superoxide radical formed during the nitrite reduction in the ETC are likely to be enough for this.

That is why the Warburg anti-effect is observed at high concentrations of CO₂ [7] [23], when the photosynthesis becomes more intense particularly at high concentrations of oxygen, as the intense glycol metabolism eventually is involved in the intensification of the function of the Calvin cycle and the formation of sucrose. Such results were obtained [23], when the atmospheric composition in the leaf chamber was changed for the wheat leaf during record of ¹⁴CO₂ only (2 min). All this indicates that, at a high photosynthesis intensity in the steady state, the glycolate cycle is completely closed to the Calvin cycle and promotes the main photosynthetic flow of carbon to the synthesis of sugars. Since the glycolate pathway is closed to the Calvin cycle, it can circulate at a high rate exceeding the carbon delivery from the outside. At the ambient concentration of carbon dioxide (0.03%), the Warburg anti-effect was observed only in plants fertilized with nitrates [23], when superoxide radical is formed during the nitrite reduction in chloroplast ETC, and the photosynthetic product export from the leaf is partially carried out in the form of amino acids. This contributes to the partial non-return of carbon to the Calvin cycle and, accordingly, the increase of the photosynthesis intensity.

It should be noted that the phosphoglycolate is the only oxidized compound that comes out of the chloroplasts into the cytoplasm in a mass flow. During its further metabolism, when glycine, serine, oxypyruvate and pyruvate are formed, all of them are concentrated in the cytosol. Therefore, when the PGA reduction to triose in the chloroplasts and its accumulation in the chloroplasts are complicated, the transfer of pyruvate to the chloroplast is blocked. As a result, all these acids are accumulated in the cytoplasm. And this is likely a very sensitive regulatory reaction that reacts to the accumulation of organic acids in the cytoplasm, with pH control of its aqueous medium.

Since the integrated flow of glycolate into the cytoplasm is 20% - 50% of the photosynthesis, it is too much to allow its accumulation within the photosynthetic cell, so after filling the vacuoles with acids, the extracellular space of the leaf is acidified as well. In turn, this activates the cell-wall invertase that hydrolyses the sucrose. With the sucrose hydrolysis, the osmolarity of the apoplastic fluid is doubled, since two molecules of hexose (glucose and fructose) are formed from one molecule of sucrose. The increased osmolarity of the apoplastic fluid as it moves to the stomatal pore and the evaporation of water increases even more (Polyakov, Karpushkin, 1981), that, in turn, osmotically closes the stomata.

As a result, the feedback of the carbon metabolism of photosynthesis with the light reactions of chloroplasts is carried out, and the formation of the number of photosynthesis products is brought into line with the request from the acceptor organs. When inhibiting the assimilate outflow from the leaf or increasing the level of nitrate supply, the glycolate pathway occurs more open. A part of the glycolate pathway intermediates (in the form of amino acids) can be exported from the leaf through the phloem to the acceptor organs, activating or generating (as with enhanced nitrate nutrition [17] [18] their metabolism in new emerging organs.

Another important indicator of changes in PCM and export of leaf photosynthetic products is the ratio of ¹⁴C labeled sucrose to hexoses (glucose and fructose). At any decrease in the intensity of photosynthesis and export of leaf assimilates, the ratio of ¹⁴C labeled sucrose to hexoses decreased. For the photosynthetic transport compound (sucrose) to be loaded into the phloem, it must leave the mesophyll cells for the extracellular space of the leaf (apoplast), where it will be loaded into the leaf phloem vessels by specific energy-dependent carrier proteins. But in the apoplast there is an enzyme, invertase, hydrolyzing sucrose to glucose and fructose [9]. The optimum activity of invertase is in the acidic pH range [9]. By changing the pH of the extracellular fluid, it is apparently possible to influence the process of sucrose hydrolysis in the apoplast, and hence it’s export to the plant sink organs.

We tested this working hypothesis in special experiments. Bean plants grown in small plastic vessels were placed under a glass bell jar, as shown in Figure 8, where either a solution of hydrochloric acid or ammonia was placed on a Petri dish. Water served as control. NH₃ dissolves in the leaf apoplastic water, binds H + ions and causes rapid alkalinization as a direct result of NH⁴⁺ formation [25].

As it was shown by experiments, acidification of the medium inhibited photosynthesis, and labeled assimilates accumulated in the apoplast. Alkalinization led to the opposite effects (Figure 8). The opposite effects of HCl and NH₃ vapors on photosynthesis and ¹⁴C content in the apoplast suggest that the key factor for the sucrose loading into phloem terminals is the pH of the apoplast

medium, which affects the activity of invertase.

Stimulation of photosynthesis under the NH₃ treatment is impossible without the successful export of assimilates from the leaf. Intensive synthesis of sucrose in the leaf must inevitably lead to its accumulation in the apoplast. However, this did not happen. It means that this intermediate compartment was successfully passed by the assimilates. Accordingly, with two-fold inhibited photosynthesis (under the action of HCl), when there is a shortage of assimilates, their accumulation in the apoplast is difficult to explain without recognizing the fact of a decrease in the sucrose export from the leaf through the phloem.

It is characteristic that in this experiment, the acidity of the extracellular medium drastically changed the ratio of labeled products of photosynthesis [16]. Excessive concentration of HCl (from an acidified solution used to displace ¹⁴CO₂ from NaH₁₄CO₃ in a gas-holder), caused [26] a decrease in the ¹⁴C incorporation into sucrose and an increase in the label content in hexoses (Table 1). By the way, a decrease (by acidification of the gaseous environment around the leaf) of ¹⁴C in phosphoric esters of sugars (PES) suggests that their conversion to sucrose was not suppressed. The formation of other labeled compounds also changes only a little. All this suggests that the main changes in the incorporation of labeled carbon into sucrose and hexose occurred as a result of stimulation of invertase and hydrolysis of sucrose in the apoplast.

To check in vivo whether the activity of the invertase enzyme itself affects PCM, we investigated genetically transformed plants with decreased or increased activity of apoplastic invertase. Experiments with potato plants, in which an additional gene for yeast apoplastic invertase was introduced, have shown [27] that additional invertase significantly reduces photosynthesis and the sucrose to hexoses ratio (especially under limiting light). Test-tube transformed plants rooted worse and, due to the shortage of photosynthetic products in the roots, were more likely to die when they were planted in the soil.

On the contrary, experiments with tomato plants (Table 2), in which the expression of apoplastic invertase in leaves was suppressed using RNA interference, showed [28] that invertase suppression increases photosynthesis (measured

using ¹⁴CO₂) by 30% - 40%, but only in young plants, which have not yet exhausted the mineral nutrition fund in their pots. Two months later, when the mineral nutrition in the pot ended, the advantages of the transformant turned into a disadvantage. The photosynthesis of plants during this time decreased two- to three-fold, but it was 25% lower in transformed plants than in control plants.

From this it follows that apoplastic invertase is involved in the regulation of photosynthesis in the whole plant system, but to influence its activity, a regulatory change in the pH of the extracellular fluid is necessary. The question arises, how does this happen?

This became clear after gasometric studies. The turning point in understanding of the mechanism of photosynthesis regulation and the role of photorespiration in this regulation was the simultaneous measurement of water vapor and CO₂ gas exchange in tomato plants under changing light [28].

In plants grown in direct sunlight, photosynthesis and transpiration were measured simultaneously using a gasometric system Walz GFS-300 (Germany), which allowed calculation of the stomatal conductance for CO₂ diffusion. The results of the experiments showed [28] that, in the steady state, photosynthesis of transformants was only 9% higher than that of the wild type (Table 3). At the same time, transpiration and stomatal conductance did not differ significantly.

But if the illumination was reduced by half (using gauze), the photosynthesis of Lin8-RNAi plants decreased to a lesser extent (and was 16% higher than that of the wild type plants). It is noteworthy that after reducing the illumination, leaf transpiration in the wild type tomato decreased significantly (by 14%), while in the transformant it increased (by 13%). As a result, stomatal conductance in the

wild type decreased (by 16%), and in transformed plants increased (by 24%). A similar connection between the stomata and mesophyll chloroplasts was also shown in potato plants [29] and later in maize plants [30] under a rapid change in illumination.

Thus, after a decrease in light stomata closed in the wild type tomato plants (as it usually happens in all plants), but became even more opened in the transformant Lin8-RNAi. From this it follows that with a decrease in illumination, the reduction of the CO₂ fixation product (phosphoglyceric acid) to sugars decreased. Unreduced acids began to accumulate first in chloroplasts, and then in the cytosol and vacuole. Then an increase in the concentration of acids occurs in the apoplast, which activates the invertase. Increased hydrolysis of sucrose in the extracellular space increases the osmolarity of the aqueous medium (two molecules of hexoses are formed from one molecule of sucrose). The solution with increased osmolarity moves towards the stomata and due to the evaporation of water its concentration (and osmolarity) increases even more. And stomata close, leading to a decrease in transpiration and photosynthesis. This happens in wild-type tomato plants.

In the transformed plants, invertase is suppressed, and sucrose is not hydrolyzed. Under these conditions, the loading of sucrose into the phloem continues, but its formation (due to a decrease in illumination) decreases. As a result, the concentration of sucrose in the extracellular solution is reduced. The osmolarity of the aqueous environment around the stomata is reduced, and the turgor of the stomatal guard cells increases. And in this case, they open even more. Accordingly, the leaf photosynthesis in Lin8-RNAi tomato plants became higher compared to the wild type plants after a decrease in illumination, since CO₂ diffusion into the leaf, and therefore its absorption increased.