Valery Yu. Lyubimov

Institute of Basic Problems of Biology RAS, Pushchino, Moscow Region, 142292 Russia.

Key words: maize, phosphoenole pyruvate carboxylase, photosynthesis, UV-radiation.

Investigations were carried out using maize (Zea mayse L.). The attached leaf of three-week-old plant was irradiated with different doses of narrow-band ultraviolet radiation (lmax= 302 nm). After that, plants were exposed under white light continuously during 2 days. During this period PEP-carboxylase activity of total protein extract (TPE) of a leaf and of purified enzyme preparation (PEP) was measured several times in irradiated and untreated leaves. Experimental results had shown that in the course of photoreactivation carboxylase activity in both TPE and PEP had displayed significant changes having oscillatory shape. Three hours after low-dose UV-irradiation slight inhibition of enzyme activity was observed in TPE preparations (20% below the control) and in PEP 20% activation of carboxylase activity was occur. After ten to twelve hours of photoreactivation significant increasing of PEP-carboxylase activity was occurred in both preparations. Peaks location was constant and did not depend on dose of irradiation. Doses of irradiation had effect on peak amplitude only. After twenty-four hours of photoreactivation oscillation of carboxylase activity began to fade, and 40-45 hours after irradiation enzyme activity reached the control level. The influence of UV-treatment of a leaf on PEP-carboxylase activity it seemed consists of two components: close-to-molecular damaged level and subcellular regulatory level.
Last two decades mechanisms of UV-irradiation effects on different vital functions of plants were investigated using new methods and ideas. Influence of UV on primary photosynthetic reactions [1,2], on structure of photosynthetic membranes [3], on individual protein components of membranes [4], on enzymes of carbon metabolism [5,6], on growth and productivity of whole plant [7] was studied.
In most papers destructive effects of ultraviolet upon different physiological functions of plant are reported. Only a few investigations demonstrate the absence of any effect of UV-irradiation [8]. Majority of known us studies was carried out using long-term irradiation with wide-band ultraviolet (zones A and B). Treatment duration usually was from several hours to several days. In such experiments I believe it may be measured cumulative effect of UV-irradiation only, and it is difficult to distinguish simultaneous (parallel) reactions of plant on UV-treatment.
I had proposed to view a long-term irradiation as superposition of short-term ones. Because the investigation of influence of minute exposures with powerful ultraviolet on PEP-carboxylase of maize leaf was undertaken.

Materials and methods.
Plant In experiments were used three-week-old plants of maize (Zea mayse L.) variety TOSS-283 (Samarskij NIISKh). Plants grown in plastic pot in the mixture of turf and sand (2:1) under illumination with 130-150 W/m2, and light spectrum from 400 nm and more. Till the UV- treatment plants grown under photoperiod 14/10 (light/dark).

 UV-treatment Second attached leaves were treated with narrow-band ultraviolet radiation cutted out by iterference filter with maximum at 302 nm. Treatment was made using intensity 10 W/m2 at three different duration: 2, 5 and 10 minutes. After UV-irradiation plants were exposed under continuous white light during 48 hours. In this period in certain time points leaf samples from control and treated plants were used for preparation of leaf extract and partially purified PEP-carboxylase.

 Enzyme preparation and measurement of activity The homogenate was prepared using middle part of second leaf in Tris-HCl (pH 7.3) buffer containing appropriate components. After centrifugation of homogenate 60 minutes at 10,000 g the resulting protein extract was divided in two parts in one of which PEP-carboxylase activity was measured. Other part of extract we used for purification of PEP-carboxylase by sedimentation of protein with 15% polyethylenglycol and chromatography on hydroxylapatite column. Hydroxylapatite was prepared according Mazin [9].PEP-carboxylase activity in both kinds of preparations was measured radiometrically. Protein was measured with Coomassie method [10].

In our experiments we used rather high intensity of incident UVB-radiation - 10 W/m2 on the level of leaf surface. It is known that natural UVB-intensity in central Europe, for example, varies between 1.0 and 2.0 W/m2 [11]. On the other hand, there are data [12] showing that more than 90% of incident UV-radiation is absorbed and reflected by leaf epidermis. Because we had the aim to investigate biochemical processes which take place in mesophyll cells the high intensity of irradiation was applyed.
In both enzyme preparations, which were prepared immediately after low dose UV-irradiation, PEP-carboxylase activity was unchanged: Fig.1 PEP-C activity after 2 min UV-irradiation.Three hours later antiphase changes of carboxylase activity of enzyme preparations were registered. The enzyme activity of TPE drops on 15-20% below of control while PEP shows the increasing of carboxylase activity up to 120%. During the period of photoreactivation of plants from 3d to 12th hours the activity of TPE grows up and achieves 130% of control level. After that PEP-carboxylase activities of preparations draw together and rich the control level 48 h after UV-irradiation.
Irradiation of leaves with "middle" UV dose Fig.2 PEP-C activity after 5 min UV-irradiation leads to the same qualitative changes of PEP-carboxylase activity as the treatment with low radiation dose. However, in first time point the activity of PEP is inhibited by 35%. Next 12 hours we observed gradual increasing of PEP-carboxylase activity in this preparation. In both TPE and PEP enzyme activity rich maximum value at the same time - 12 hours after UV-treatment. These maximal activities numerically were the same as after low dose irradiation. Both curves achieve the control level at 48's hour of photoreactivation, too.
Most powerful dose of UV-irradiation of maize leaves produces significant changes in postirradiation dynamics of PEP-carboxylase activity: Fig.3 PEP-C activity after 10 min UV-irradiation. Initial activity of PEP dropped up to 30% of control. Further photoreactivation leads to increasing of activity to control level only, without exceeding them. Inhibition of enzyme in TPE between 3d and 6th hours become stronger, too. And maximal activity which is richen at 12-15th hour not significantly higher than control. Nevertheless, PEP-carboxylase activity in the TPE preparation achieves control level at 48th hour and in the PEP much earlier.

Principle features of our experiments are:
Short-term, powerful, narrow-band Fig.4 Transmittance spectrum of interference filter UV-irradiation of attached leaves in the absence of visible light.
Following permanent illumination of plants by light similar to sunlight.
Measurement of PEP-carboxylase activity in total protein extract of a leaf as well as in purified enzyme preparation

Post UV-irradiation dynamics of PEP-carboxylase activity it is obvious consists of three phases. First phase in both kinds of preparation is the inhibition of enzyme activity. At that, the activity of purified enzyme at zero time is not affected by low dose UV-treatment, and at higher doses dramatically slows down. Minimal activity in TPE is significant at highest UV dose only, and in all cases it delays by 3-5 hours in compare with PEP. It shows higher stability of PEP-carboxylase in presence of cellular compounds than in purified form. It is co-ordinated with enzyme activities in second phase - phase of activation. In this period (approx. 12 h after UV-treatment) the activity of TPE is higher than of PEP, too. It may be marked that at low dose of UV maximal activity in TPE delays in compare with one in PEP. At highest UV dose the maximal carboxylase rate statistically not exceed the control level. In the third phase (24-48 hours) the riching of enzyme activities of control level occurs.

Perhaps dynamics of PEP-carboxylase activity during photoreactivation of UV-treated leaves of maize reflects two independent processes. One of them is destructive effect of UV leading to damage of enzyme molecules immediately after irradiation. In the course of subsequent illumination it seem the renewal of enzyme pool takes place. Another process is suggested by higher activity of enzyme in TPE preparation and consists, perhaps, of production of any compounds stubilizing enzyme(s). Such two-component mechanism of enzyme response on UV-treatment corresponds to non-specific stress reaction of organisms and is confirmed by three-phase dynamics. At last, really, if we consider a reaction of photosynthetic apparatus of plant on prolonged UV-irradiation as superposition of series of short treatments we can see that photosynthetic rate can either decrease or increase depending on UV characteristics and plant species [13].

Literature cited.

  1. Gordenova OS, Veselova TV, Veselovsky VA, Gun-Azhav T Influence of ultraviolet radiation on primary photosynthetic reactions of wheat leaves // Biol. nauki. 1988, issue 11, p. 27. [in Russian]
  2. Brandle JR, Campbell WF, Sisson WB and Caldwell MM (1977) Net photosynthesis, electron transport capacity and ultrastructure of Pisum sativum L. exposed to ultraviolet-B radiation. Plant Physiol 60:165-169
  3. Khalilov RI, Khomutov GB, Tikhonov AN Influence of ultraviolet radiation on structural-functional features of thylakoid membranes //Fiziol. rast. 1993, .40, N 3, . 373-378. [in Russian]
  4. Greenberg B.M., Gaba V., Gannani O., Malkin S., Mattoo A.K., Edelman M. Separate photosensitizers mediate degradation of the 32 kDa photosystem II reaction center protein in the visible and UV spectral regions // Proc. Natl. Acad. Sci. USA, 1989. V.86. P. 6617-6620.
  5. Vu C.V., Allen L.H., Garrard L.A. Effect of supplemental UV-B radiation on prymary photosynthetic carboxylating enzymes and soluble proteins in leaves of C3 and C4 crop plants // Physiol. plant., 1982, v.55, p.11-16.
  6. Vu CV, Allen LH and Garrard LA Effects of enhanced UV-B radiation (280-320 nm) on ribulose-1,5-bisphosphate carboxylase in pea and soybean // Environ Exp Bot, 1983, v.24, p.131-143
  7. Teramura A.H. Effects of ultraviolet-B radiation on the growth and yield of crop plants // Physiol. plant., 1983, v.58, p.415-427.
  8. Beyschlag W., Barnes P.W., Flint S.D., Caldwell M.M. Enchanced UV-B irradiation has no effect on photosynthetic characteritics of wheat (Triticum aestivum L.) and wild oat (Avena fatua L.) under greenhouse and field conditions // Photosyntetica, 1988, vol. 22, p.516-525.
  9. Mazin A.L., Sulimova G.E., Vanyushin B.F. Granulated hydroxylapatite: preparation and chromatographic properties // Anal. Biochem., 1974, v.61, p.62-71.
  10. Bradford M.M. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding // Anal. Biochem., 1976, v.72, p.248-254.
  11. Internet ELDONet Project: http://www.ib.pi.cnr.it/eldonet/index.html
  12. Shul'gin I.A. Plant and Sun // L.: Hydrometheoizdat, 1973, .251
  13. Lyubimov V.Yu., Kalevich A.E., Somansurov S. Influence of reduction of enhanced mountain UV-radiation on the photosynthesis and growth of cultured C3-plants // 1st Internet Conf. Photochem. Photobiol., http://www.photobiology.com/v1/lyubimov/

Thank you for reading this poster