METHOD OF INCREASING OF THE PHOTOSYNTHETIC CARBON DIOXIDE ASSIMILATION YIELD

Technical field of invention

This invention deals with a method of increasing the yield of photosynthetic assimilation of carbon dioxide (CO ₂ ) in plants by means of application of agents that increase the carboxylase activity of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39).

Background of the invention

Probably the most abundant protein of the biosphere on Earth, the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39) is the only enzyme able to prepare biomass in plants from inorganic compounds. It is present in high concentration in chloroplasts and represents up to 50 % of the soluble leaf proteins. During photosynthesis the energy of light is transformed to chemical energy, oxygen is released from water and the gaseous carbon dioxide from the air is fixed into organic compounds. In consequent steps are formed sugars, amino acids, fatty acids and other parts of living mass. RuBisCO is a unique and very interesting enzyme. It can be distinguished as the most important enzyme within the photosynthesis, catalyzing the key reaction of the Calvin cycle, so called carboxylation, during which the CO ₂ is built into organic molecules. The enzyme RuBisCO is responsible for transformation of inorganic carbon into organic compounds in case of so called C3- plants, which represent 90 % of all 300 000 until now known plant species, the residual 10 % of plants has in addition to RuBisCO also other alternative enzyme pathways serving for fixation of carbon dioxide (CO ₂ ), but these pathways serve only to the purpose of increasing the concentration of CO ₂ in the neighbourhood of the enzyme RuBisCO. RuBisCO forms within the Calvin cycle 3-phosphoglycerate. From 3-phosphoglycerate is formed glyceraldehyde-3 -phosphate, serving as starting compound for further formation of glucose, starch and other compounds (Malkin R, Niyogi K. Photosynthesis. In book: Biochemistry and Molecular Biology of Plants. Buchanan B.B., Gruissem W., Jones R.L. (Eds.), American Society of Plant Physiologists, Rockville, MD, USA, chapter 12, 568-628 (2000); Raghavendra A. S. Photosynthesis and partitioning in C3 plants. In book: Encyclopedia of

Applied Plant Science. Thomas B., Murphy D.J., Murray B. G., (Eds.), Elsevier, Oxford, 673-680 (2003)).

For the reasons mentioned above, changes in activity of the enzyme as well as in its content are of importance. Plant conversion of inorganic carbon into organic matter is very important for global cycling of carbon. The capacity and the yield of photosynthesis are influenced by illumination, temperature, and the concentration of available CO ₂ . Except for the ability to bind CO ₂ , RuBisCO acts as oxygenase, too, which rather decreases the photosynthesis yield but it enables the plant to synthesize glycine and serine. The ratio of those activities is controlled by the ration of partial pressures of CO ₂ and O ₂ .

RuBisCO is a complex protein made up of 8 large subunits (about 50 000 Da each) and 8 small subunits (about 14 000 Da each). The large subunit is encoded for in the plastid genome, whereas the small subunit in the nucleus. Within photosynthesis, the solar energy is converted into chemical energy represented by molecules of NADPH and ATP. During CO ₂ fixation by RuBisCO, both these compounds are consumed.

Photosynthesis in C3-plants is regulated in many ways. For instance, plant hormones, such as brassinosteroids, are known to positively influence the photosynthesis yield (Hradecka, Kohout & Hold, Kohout - unpublished results). During photosynthesis the energy of light is converted into chemical energy in the form of NADPH and ATP. During CO ₂ fixation by the enzyme RuBisCO within the Calvin cycle these compounds are consumed. For the enzyme to work properly, Mg ²⁺ ions need to be available. The activity of RuBisCO and its turn-over influence CO ₂ fixation efficiency; for instance, RuBisCO level in plants decreases with the lack of nitrogen and thus the photosynthetic fixation of CO ₂ is limited. RuBisCO is inactive in the dark; it requires illumination to become active. The activation is a result of carbamylation, which consists of binding of CO ₂ and Mg ²⁺ onto lysine residue in the active centre closeness. The carbamylation changes the conformation of the large subunit and activates the enzyme while Mg2+ stabilizes the active conformation. RuBisCO is thus only active when lysine 201 reacts with CO ₂ in the active centre nearness, allowing Mg ²⁺ to bind. Noncarbamylated enzyme binds ribulose 1,5-bisphosphate (RuBP) too tightly for the catalytic reaction to occur. However, RuBisCO can be inhibited by some compounds such as 2-carboxyarabinitol-l -phosphate (CAlP).

Another protein, RuBisCO activase, releases inhibitors (e.g. CAlP) that bind to the active site. The inhibitors hinder the activation (carbamylation) of the enzyme in the dark. RuBisCO activase is activated by light by means of ATP. The activation of RuBisCO and its activase by light represent the next mechanism of important light regulation of photosynthetic CO ₂ assimilation.

CAlP, a compound naturally present in the leaves of many plants, is a strong inhibitor of RuBisCO. The affinity of the enzyme to CAlP is much higher than to the substrate. CAlP accumulates in the dark and blocks the active sites, and is released after illumination or after RuBisCO activase action. However, it was not found in all plants. RuBisCO and other cooperating enzymes are activated by the increase of pH and Mg ²⁺ concentration; they are almost inactive at pH 7.2.

The significance of influencing RuBisCO activity consists in the ability to increase the yield of photosynthesis. Most agriculture crops (cereals, legumes, oil plants) belong to plants with C3 metabolism. Three main factors limit the photosynthetic yield; losses by photorespiration (which is an unavoidable result of oxygenase activity of RuBisCO), high water requirements, and temperate zone preference. A known factor that increases the efficiency of photosynthetic CO ₂ fixation is an increased level of CO ₂ . RuBisCO is a rather slow-working enzyme; one molecule is able to fix only three molecules of CO ₂ per second, thus each increase of reaction rate is significant in global effectivity of the process.

Thus influencing the enzyme in the way that would lead to a higher activity is of particular significance for the increase in the photosynthetic yield, which is important primarily in the agriculture, as well as for the increase in the fixation of atmospheric CO ₂ , hence in fighting high CO ₂ concentrations in the atmosphere.

Summary of the invention

Inventors of this invention have found out that some low-molecular organic compounds influence the activity of RuBisCO. Under standard conditions, it has been discovered that these compounds increase the CO ₂ fixation by RuBisCO in vitro by more than 10% on average. It also represents increase in organic matter formation alike as well as significant fixation of CO ₂ from the atmosphere.

As an example, ecdysteroids can be stated. Ecdysteroids belong to plant oxysterols whose hormonal effect on insects had been proved. Their function in plants has been unknown yet. We discovered that these compounds influence the carboxylase activity of the enzyme. The active enzyme consists of 8 large subunits and 8 small subunits considered to have regulatory role. We identified the small subunits as proteins able to bind 20- hydroxyecdysone (20HE) in bioaffinity chromatography. Further testing revealed the affinity of related oxysterols to both subunits of RuBisCO.

In a similar way, also other oxysterols act. These include brassinosteroids, plant hormones that influence plant growth and particularly are involved in suppressing stress response in plants.

When assaying carboxylase activity of RuBisCO with the use of ¹⁴ C-labeled CO ₂ , molar ratio of the steroid to enzyme 1 :1 did not show significant increase in activity. However, when the molar ratio was 10:1, the increase in activity is higher than 10%. When twenty fold excess of steroid is applied, no further increase appears.

After analytical assay was optimized and the hypothesis has been verified that 20- hydroxyecdysone is able to influence the carboxylase activity of RuBisCO, screening of available steroids and other compounds was carried out in order to find out whether they are able to influence the carboxylase activity of RuBisCo as well.

Examples

Example 1

Effect of 20-hydroxyecdysone (20E) on the yield of RuBisCO carboxylase reaction.

Relative amount of product detected represents the ratio of the amount of [l-14C]-3- phosphoglycerate produced by the reaction in the presence of 2OE to the amount of [1-14C]- 3-phosphoglycerate produced by the reaction in no presence of 2OE.

The influence of ecdysteroids on RuBisCO assay was based on the protocol described by Pierce et al. (Pierce JW, McCurry SD, Mulligan RM, Tolbert NE. Activation and assay of

ribulose-l,5-bisphosphate carboxylase/oxygenase. Methods Enzymol. 1982; 89: 47-55.), where the RuBisCO carboxylase activity is assayed by the rate of formation of acid-stable ¹⁴ C product, [l- ¹⁴ C]-3-phosphoglycerate, formed from NaH ¹⁴ CO ₃ . First, the enzyme from spinach was activated at 2 mg.mL ^"1 in the activating buffer - 100 mM Bicine pH 8.2, non-labelled 10 mM NaHCO ₃ , 20 mM MgCl ₂ , 0.2 mM EDTA and 1 mM dithiothreitol - for ten minutes at 30 ⁰ C. The assay itself was carried out in 20 ml scintillation vials in a solution prepared by mixing 250 μL of 200 mM bicine buffer pH 8.2, 0.4 mM EDTA and 1 mM dithiothreitol, 5 μL of 2 M MgCl ₂ , 150 μL of 2OE solution or water (in the case of the control), 20 μL of 25O mM NaH ¹⁴ CO ₃ solution and 20 μL of 12.5 mM ribulose-l,5-bisphosphate. 25O mM NaH ⁴ CO ₃ was prepared by mixing NaH ¹⁴ CO ₃ solution (specific activity 2 mCi/ml) with 0.3 M NaHCO ₃ in the ratio of 1 :4. The reaction was initiated by the addition of 100 μL of the activated enzyme and the vial was swirled so that the solution components could mix. The reaction was stopped by the addition of 200 μL of 2 M HCl. To remove excess ¹⁴ CO ₂ and acid, the vials were slowly heated at 95 ⁰ C in a hood. After the liquid evaporated and the vials were cooled at laboratory temperature, 5 mL of scintillation solution Rotiszint eco plus was added. The concentration of 2OE was chosen so that its final molar concentration to the molar concentration of the enzyme in the vial was 1:1; the stock solution containing 1 mg of 2OE per ml was diluted to the desired concentration just before the experiment. Results in Table 1.

Table 1

Relative amount of product detected

Without steroid 1

20-hydroxyecdysone (1:1)* 1.05

*Numbers in brackets represent the molar ratio of steroid to enzyme - 1:1 means that 1 mg of the enzyme was in the mixture with 8,75.1C ⁴ mg of 20-hydroxyecdysone.

Example 2

Relative amount of product detected in the presence of different amounts of 2OE to the amount in no presence of 2OE.

The same experiment as in Example 1 was carried out but different amounts of 20- hydroxyecdysone were used. Results in Table 2.

Table 2

Relative amount of product detected

without steroid 1.00

20-hydroxyecdysone (1 :1)* (see example 1) 1.05

20-hydroxyecdysone (10:1)* 1.13

20-hydroxyecdysone (20:1)* 1.12

* Numbers in brackets represent the molar ratio of steroid to enzyme - 10:1 means that lOfold excess of steroid over RuBisCO was used.

Results in Table 2 show that lOfold increase in ecdysteroid amount resulted in the increase of carboxylase activity. However, further increase did not show further increase in the activity of the enzyme.

Example 3

Relative amount of product detected in the presence of different brassinosteroids to the amount in no presence of them.

The same experiment as in Example 1 was carried out but different brassinosteroids were used. Results in Table 3.

Table 3

Relative amount of product detected

Without steroid 1.00

24-epibrassinolide (10:1) 1.10

24-epicastasterone (10:l) 1.20

Example 4

Relative amount of product detected in the presence of different ecdysteroids to the amount in no presence of them.

The same experiment as in Example 1 was carried out but different ecdysteroids were used. Results in Table 4.

Table 4

Relative amount of product detected

Without steroid 1 .00

Ajugasterone C (10:1) 1 .13

Polypodine B (10:l) 1 .11

Example 5

Relative amount of product detected in the presence of mixed ecdysteroids to the amount in no presence of them.

The same experiment as in Example 1 was carried out but mixed ecdysteroid fractions were used. Results in Table 5.

Table 5

Relative amount of product detected

Without steroid 1.00

80% 20-hydroxyecdysone (10:1) 1.11

Ecdysteroidal fraction from root extracts of Leuzea carthamoides 1.13

Example 6

Steroids influence on overall activity of photosynthesis in vivo

Activity of photosynthesis was measured as the production of oxygen by circular target (average 35 mm) cut from the third leaf (counted from the top of the plant) that was placed in the sampling chamber. Assay: oxygen electrode (Clark electrode), sampling chamber LD2 (Hansatech, King's Lynn, Great Britain). Target was incubated in the dark for 5 minutes followed by 9 minutes of illumination; production of oxygen evaluated in the fifth-eighth minute after illumination (in the linear part of the light curve). Illumination was 240 micromol.mλs ^'1 . Plants were 96 days old when analyzed (96 days after sowing). The ratio of 2OE to enzyme was 1 :10, thus the most effective from Example 2. Results are presented in Table 6 and Figure 1.

Table 6

Net photosynthetic rate. PN (μmol O ₂ .m ^"2 .s ^"1 )

Table 6 as well as Figure 1 show that statistically evidential differences were found 4 and 6 hours after treatment. Less evidential difference was achieved after 1 hour.

When monitoring steroids effect, statistically evidential differences decrease, however, increase in activity dies down after 50 hours.

Industrial applicability

Applicability of the invention lies in the increase of green matter (due to increase in carboxylase activity of the enzyme) as well as in the increase of CO ₂ assimilation, which actually could be even more important. Thus the invention can be considered as an effective means of greenhouse gases fighting as 10% increase in the activity of the enzyme entails great contribution in CO ₂ decrease from the atmosphere.