ISSN 0003-6838, Applied Biochemistry and Microbiology, 2007, Vol. 43, No. 6, pp. 635–640. © Pleiades Publishing, Inc., 2007. Original Russian Text © N.I. Nen’ko, V.K. Plotnikov, N.A. Kuzembaeva, V.N. Garazha, E.V. Surkova, A.I. Nasonov, Yu.S. Pospelova, N.G. Malyuga, 2007, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 6, pp. 715–721.
The Effect of Furolan on the Physiological and Biochemical Characteristics of Ripening Winter Wheat Grain N. I. Nen’koa, V. K. Plotnikova, b, N. A. Kuzembaevab, V. N. Garazhaa, E. V. Surkovaa, A. I. Nasonovb, Yu. S. Pospelovaa, and N. G. Malyugac a
b
Kuban State Technological University, Krasnodar, 350072 Russia Luk’yanenko Krasnodar Institute of Agriculture, Krasnodar, 350012 Russia e-mail:
[email protected] c Kuban State Agricultural University, Krasnodar, 350044 Russia Received March 23, 2006
Abstract—The effects of the preparation Furolan, (2-furyl-2)-1,3-dioxolane, on the degree of mRNA polyadenylation and the pattern of protein synthesis in the ripening grain of several soft winter wheat (Triticum aestivum L.) cultivars were studied. It was demonstrated that Furolan stabilized mRNA in a cultivar-specific manner, thereby accelerating to various degrees the biochemical processes taking place in the ripening grain. Of the wheat cultivars studied, Krasnodarskaya 99 was the most responsive cultivar with respect to a set of changes in nucleic–protein metabolism; the cultivar Deya was next followed by the cultivar Bat’ko. The cultivar Kroshka did not respond to the treatment with Furolan. The cultivar specificity of this preparation allows its practical application to be optimized. DOI: 10.1134/S0003683807060129
Improvement of grain quality is the most important problem in wheat grain production. The grain during ripening is exposed to various adverse factors, which deteriorate its milling and bread-making properties. Breeding is an extremely long process and sometimes fails to produce a cultivar displaying high adaptive and technological characteristics. However, efficient technological practice assists plants in resisting the adverse environmental conditions, for example, application of growth regulators with an antistress effect (adaptogens), allowing the cultivars to fuller realize their genetic potential. Of special interest are the substances stimulating the organism resistance by activating the molecular mechanisms of adaptation that are idle under normal conditions. Unfortunately, the arsenal of such adaptogenic agents is still very poor. Along with an empiric search, an expansion of this arsenal requires, in particular, to design purposefully the preparations that would favorably act on various components of cell metabolism. The preparation Furolan, (2-furyl-2)-1,3-dioxolane, was synthesized using furfural at the Kuban State Technological University. It has undergone the state trials in wheat, is ecologically pure, and has been approved for application on the territory of Russia. The field trials of the preparation Furolan, conducted in 1990–2003 with various cultivars of winter wheat grown in Krasnodar krai, have demonstrated that treatment of crops increases the grain yield by 10–15%. The preparation slows down the growth of culm internodes 1–3 under favorable growth conditions and allows the plants to
retain a high activity of growth and synthetic processes in the case of drought [1]. It was of doubtless interest from both theoretical and practical standpoints to study the molecular grounds of Furolan action on the biology of winter wheat. One of the most well-grounded hypotheses concerning the action of adaptogens is the assumption that they are similar to weak irritants (stressors), i.e., make very small injuries to the organism, thereby enhancing the mobilization of its defense forces [2, 3]. As is known, biologically active substances can influence all of the stages of realization of genetic information, from DNA replication to protein synthesis [4]. The most efficient mechanisms involved in the protein synthesis of eukaryotes are the posttranscriptional changes in mRNA. The most labile of them is the change in mRNA stability: the more stable mRNA is, the wider possibilities exist for protein synthesis and, vice versa, a decrease in mRNA stability hampers protein synthesis [5–7]. We have earlier demonstrated that well-studied biologically active substances, such as gibberellic acid, cyclic adenosine monophosphate, and sodium salicylate, regulate plant metabolism, in particular, via changing the stability of mRNA [8]. The goal of this work was to study the effect of Furolan on the nucleic and protein metabolism in ripening grain of various winter wheat cultivars and the action of Furolan on mRNA stability in the ripening grain as the central component of the system implementing the genotype–medium interactions and to analyze the changes in protein composition.
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Table 1. The effect of Furolan on the nucleic acid content in the ripening grain of soft winter wheat cultivar Krasnodarskaya 99, µg/ml Ripening stage Milk ripeness Wax ripeness
RNA
DNA
RNA/DNA
Control
Furolan
Control
Furolan
Control
Furolan
0.64 ± 0.01 0.47 ± 0.04
0.52 ± 0.01 0.68 ± 0.02
0.21 ± 0.01 0.25 ± 0.03
0.25 ± 0.01 0.37 ± 0.02
3.04 1.88
2.08 1.84
MATERIALS AND METHODS The experiments were conducted with the ripening grain of soft winter wheat (Triticum aestivum L.) under field conditions during the summers of 2003 and 2004. Furolan was applied at the early booting stage at a dose of 4 g/ha. The following cultivars bred at the Luk’yanenko Krasnodar Institute of Agriculture and differing in the genetically determined response to biologically active substances were used in the work: Deya, Krasnodarskaya 99, Kroshka, and Bat’ko. Grain was collected at the stages of milk and wax ripeness and stored in liquid nitrogen. For RNA isolation, wheat grain frozen in liquid nitrogen was ground to a dust state to extract RNA with the buffer containing 200 mM Tris–HCl pH 8.5, 250 mM sucrose, and 50 mM MgCl2 at a ratio of sample to buffer of 1 : 5 (wt/vol). Starch was removed by a short centrifugation. Then the buffer was supplemented with sodium dodecylsulfate (SDS) to a final concentration of 1%. High-molecular-weight RNA was extracted with a phenol–chloroform (1 : 1) mixture and precipitated with lithium chloride and urea at a concentration of 4 M each. The ratio of RNA to DNA was determined as described in [9]. Stability index (SI) of the total poly(A)-containing mRNA was determined by affinity chromatography on poly(U) Sepharose as the ratio of poly(Ä)++ mRNA (double purification) to poly(Ä)+ mRNA (single purification). The length of terminal poly(A) sequence was studied using stepwise mRNA elution from a column filled with poly(U) Sepharose with buffer solution of various temperatures (35°ë, the mRNA with relatively short poly(A) sequences and 65°ë, with relatively long poly(A) sequences). The elution buffer contained 0.01 M Tris–HCl pH 7.5 and 0.05% SDS [8]. The fraction composition of protein was examined by a successive extraction with 70 mM phosphate buffer pH 7.0 containing 1 M NaCl, 7% ethanol, and 0.05 M NaOH according to the Osborne method [10]. The proteins extracted with 20 mM Tris– HCl buffer pH 7.4 supplemented with 0.1% β-mercaptoethanol were assayed using fractionation by gel filtration on Sephadex G-150 (column of 1 × 50 mm) and ion-exchange chromatography on DEAE cellulose (column of 1.5 × 10 cm) in a stepwise NaCl concentration gradient (0 to 1 M). The protein content in fractions was recorded at 280 nm and calculated according to the Warburg–Christian equation [11].
All calculation results are shown per unit raw weight. The tables and figures show arithmetic means obtained from three–five analytical replicates and the standard errors. The following reagents were used in the work: poly(U) Sepharose and Sephadex G-150 (Pharmacia, Sweden), DEAE cellulose (Reanal, Hungary), and βmercaptoethanol (Serva, Germany). RESULTS AND DISCUSSION The effect of Furolan on nucleic acids in ripening wheat grain. Furolan decreased the ratio of RNA to DNA in ripening grain (Table 1). This indicates a decrease in DNA functional activity; however, the protein-synthesizing activity of mRNA in this case is realized to a higher degree, as its degree of polyadenylation (the length of 3'-terminal poly(A) sequence) increases (Table 2). It is known that the degree of polyadenylation determines the stability and translational activity of mRNA: the longer 3'-terminal poly(A) sequence of mRNA are, the more stable mRNA (longer halflife) is and the higher its translational ability (the intensity of protein synthesis) [5]. Cultivar-specific distinctions in the action of Furolan were observed, namely, the degree of mRNA polyadenylation increased in the cultivar Krasnodarskaya 99 at the initial stage of grain formation but not at the stage of milk ripeness. The cultivar Bat’ko displayed an analogous increase only at milk ripeness, and no changes in the degree of polyadenylation were recorded in the cultivar Kroshka. The effect of Furolan on the degree of mRNA polyadenylation depended on the conditions of a particular year: the increase in poly(A) sequences of mRNA in the cultivar Bat’ko in 2003 was recorded at both wax and complete ripeness, whereas this was recorded already at the stage of grain formation and at milk ripeness in 2004. An increase in mRNA polyadenylation in the cultivar Kroshka was observed in neither year. In 2004, the extending of poly(A) sequence was not found in the cultivar Deya too. The effect of Furolan on the yield of high-molecular-weight RNA isolated from grain also changed depending on the conditions of a particular year. The effect of Furolan on the fraction composition of wheat grain proteins. The classification of proteins of grain crops proposed by Osborne is based on protein solubility. Successive extractions of proteins from flour with water, saline solution, alcohol, and alkali divide
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Table 2. The effect of Furolan on mRNA stability in the ripening grain of soft winter wheat (data of 2003−2004) Cultivar Kroshka (2003)
Stage Milk ripeness Wax ripeness Complete ripeness
Bat'ko (2003)
Milk ripeness Wax ripeness Complete ripeness
Bat'ko (2004)
Grain formation Milk ripeness Wax ripeness
Krasnodarskaya 99 (2004)
Grain formation Milk ripeness Wax ripeness
Deya (2004) Grain formation Milk ripeness
Variant
RNA, % of control
Control Furolan Control Furolan Control Furolan Control Furolan Control Furolan Control Furolan Control Furolan Control Furolan Control Furolan Control Furolan Control Furolan Control Furolan Control Furolan Control Furolan
100 76 100 119 100 92 100 73 100 128 100 141 100 109 100 85 100 127 100 83 100 124 100 105 100 85 100 104
them into albumins, globulins, prolamines (gliadins), and glutelins (glutenins). The boundaries in classification of the Osborne fractions are fairly conditional; however, they are most well-studied and widely used in research practice when examining the physiological and biochemical processes during grain formation and germination. Gliadins and glutenins are the major proteins involved directly in gluten formation and responsible for many technological properties of flour. The component range of these proteins is determined by the specific genetic features of particular cultivar. However, the ratio of the components within a protein fraction may essentially change depending on the weather, especially, during grain plumping and ripening. Albumin and globulin fractions contain mainly enzymes as well as constitutive nuclear and cytoplasmic proteins, including nucleoproteins, lipoproteins, etc. As is APPLIED BIOCHEMISTRY AND MICROBIOLOGY
mRNA, % of total RNA 1.7 1.7 1.6 1.4 2.0 1.8 1.9 2.0 2.5 1.2 2.2 1.9 1.8 1.9 2.8 2.6 2.5 1.2 2.0 3.6 2.7 2.6 1.2 1.1 2.0 1.3 2.0 1.9
Degree of polyadenylation, (A)n65°C/(A)n35°C 0.80 0.76 0.34 0.66 0.35 0.30 0.36 0.35 1.59 4.0 0.60 0.79 0.45 0.41 0.59 0.91 1.59 4.00 1.90 2.10 1.02 1.00 0.93 1.20 0.54 0.50 0.54 0.47
Stability index, %
78 80 55 70
74 86 67 67
92 75 77 78
reported, the gluten contains approximately half of the soluble proteins of the grain [12, 13]. The fraction compositions of ripe grain proteins of the winter wheat cultivars Kroshka, Deya, and Bat’ko were determined. The results are listed in Table 3. It is found that Furolan significantly increases the contents of alcohol-soluble (prolamines), water-soluble (albumins), and saline-soluble (globulins) fractions in the grain of cultivar Deya. As for the cultivars Bat’ko and Kroshka, only insignificant differences in the contents of these fractions were observed. The effect of Furolan on the dynamics of soluble proteins in ripening wheat grain. Gel filtration on Sephadex G-150 was used to study the effect of Furolan on the protein component composition of ripening wheat grain (Table 4). The fraction of high-molecularweight proteins (with a molecular weight of 20 to
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Table 3. The effect of Furolan on the protein fraction composition in winter wheat grain Cultivar Kroshka Bat'ko Deya
Albumins + globulins, mg/g
Prolamines, mg/g
± control, %
Control
Furolan
114.92 ± 7.58 102.12 ± 7.64 100.95 ± 5.95
113.42 ± 11.50 104.47 ± 1.41 121.00 ± 5.24
–1.3 2.0 18.9
70 kDa) increases with ripening and, correspondingly, the fraction of low-molecular-weight proteins (with a molecular weight of 5 to 10 kDa) decreases. This process accelerated at the wax-ripe stage of the Krasnodarskaya 99 grain; in the case of the cultivars Deya and Bat’ko, the distinctions were detectable already at the stage of milk ripeness. A cultivar-specific character of Furolan action is evident. The fraction of high-molecular-weight proteins obtained by gel filtration was then examined by ionexchange chromatography on DEAE cellulose. The cultivars studied displayed the changes in protein ratios: the fraction of positively charged proteins decreased (from 96 to 40%) and, correspondingly, the fraction of negatively charged proteins increased (Figs. 1 and 2). The cultivar Krasnodarskaya 99 displayed a higher variation in the protein content in the experimental variant compared with the control. The most pronounced changes were recorded for the following fractions of the eluate: 0.2, 0.3, 0.5, and 0.6 M NaCl at milk ripeness and 0.1, 0.2, 0.3, and 0.8 M NaCl at wax ripeness. In the case of the cultivar Deya, the most pronounced differences were observed at the stage of milk ripeness in the fractions eluted with 0.1, 0.2, and 0.8 M NaCl and at wax ripeness in the fractions eluted with 0.1 and 1 M NaCl. As for the cultivar Bat’ko, the maximal distinctions between the experiment and control were recorded at milk ripeness in the fraction eluted with 0.2 M NaCl and at wax ripeness in the fractions 0.2, 0.3, 0.4, 0.8, and 1 M NaCl. It has been demonstrated that Furolan accelerates biochemical processes in the ripening wheat grain in a Table 4. The ratio of (1) high- and (2) low-molecular-weight proteins in fractions of the eluate from Sephadex G-150 %) Stage
Variant
Milk ripeness Control Furolan Wax ripeness Control Furolan
Krasnodarskaya 99
Deya
Bat'ko
± control, %
Control
Furolan
23.68 ± 1.99 24.63 ± 1.67 22.30 ± 1.40
26.34 ± 0.48 26.03 ± 0.47 27.62 ± 0.90
2.4 5.2 13.0
cultivar-specific manner. Of the cultivars studied, Krasnodarskaya 99 was most responsive to the action of Furolan followed by Deya and Bat’ko. The effect of Furolan on the gluten content and quality in winter wheat grain was of the greatest interest. The results obtained are listed in Table 5. Furolan increased the content of raw gluten in the ripe grain of Krasnodarskaya 99 by 40.6% compared with the control; of the cultivar Deya, by 23.6%; and of the cultivar Bat’ko, by 25%. The activation of protein biosynthesis assisted the increase in the content and quality of raw gluten in grain. According to our data, application of Furolan to the cultivar Kroshka failed to change the quality of its grain. The treatment with Furolan did not cause any changes in the studied characteristics of nucleic acids, thereby suggesting that this cultivar has no receptors for exogenous biologically active substances. Thus, the general pattern of the changes in nucleic and protein metabolism in the ripening grain of soft winter wheat caused by Furolan demonstrates the effect of “functional reservation,” i.e., the ability of biological molecules to display a considerably higher activity than is necessary for normal cell development. As has been demonstrated earlier, the physiological stress (hardening stress zone) increases the degree of mRNA polyadenylation and, correspondingly, elevates the translational activity of plant cell polyribosomes [8]. It is known that a moderate dehydration, not only fails to decrease, but also elevates the activity of enzymes that activate amino acids; presumably, this is necessary for plant adaptation to intensified protein synthesis—induction of the synthesis of new enzymes that are necessary, in particular, to intensify the pentaTable 5. The effect of Furolan on the quality of winter wheat grain
Cultivar
Protein content, %
Raw gluten content, %
Gluten quality, GDI units
1
2
1
2
1
2
Control
Furolan
Control
Furolan
Control
Furolan
66 70 72 79
34 30 28 21
67 75 85 82
33 25 15 18
63 37 84 16 86 14 93 7
Krasnodar- 9.3 skaya 99 Deya 11.8 Bat'ko 11.8
11.2
12.8
18.0
80
60
13.4 14.2
16.5 19.2
20.4 24.0
80 75
70 70
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(a) % 18 16 14 12 10 8 6 4 2 0 % 18 16 14 12 10 8 6 4 2 0 % 25
2
I
II
III
(b) % 80 70 60 50 40 30 20 10 0
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I
II
III
(c) % 80 70 60 50 40 30 20 10 0
1
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3 4
5
8 67
2
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III
IV
(b) 1 3 4 5 67
2
8
I
II
2
III
IV
III
IV
(c) 20 15
I
II
III
5
10 Fig. 1. Changes in the content of positively charged proteins (%) in the ripening grain of the wheat cultivars (a) Krasnodarskaya 99, (b) Deya, and (c) Bat’ko (ion exchange chromatography on DEAE cellulose, elution with 20 mM Tris– HCl pH 7.4: (I) stage of grain development, (II) milk ripeness, and (III) wax ripeness; (1) control and (2) experiment (treatment with Furolan).
1
34
5
7 8
0 I
phosphate respiration pathway on the background of the weakened glycolytic pathway [14]. A mechanical injury of plant causes an elevation in the respiration rate and a de novo synthesis of many enzymes, which is determined by formation of the polyribosomes with increased translation activity. An analogous increase in the translation activity of plant polyribosomes in a cellfree system for protein synthesis (in vitro) is also observed at elevated temperatures, action of pathogens, as well as at a hardening temperature, moderate salting, and dehydration [15, 16]. Therefore, the protein-synthesizing activity of mRNA is realized to a fuller degree on the background of the decreased DNA functional activity under conditions of physiological stress. APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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II
Fig. 2. Changes in the content of negatively charged proteins (%) in the ripening grain of the wheat cultivars (a) Krasnodarskaya 99, (b) Deya, and (c) Bat’ko; ion exchange chromatography on DEAE cellulose, elution with 20 mM Tris–HCl pH 7.4 in a stepwise NaCl concentration gradient: (1) 0.1 M, (2) 0.2 M, (3) 0.3 M, (4) 0.4 M, (5) 0.5 M, (6) 0.6 M, (7) 0.8 M, and (8) 1.0 M; (I) milk ripeness, control; (II) milk ripeness, experiment (treatment with Furolan); (III) wax ripeness, control; and (IV) wax ripeness, experiment (treatment with Furolan).
Furolan changes the protein synthesis, not only quantitatively, but also qualitatively. Presumably, this determines an increase in the plant resistance to stress environmental conditions and a cultivar-specific increase in crop yield (by 10–15%). It is known that changes in isozyme composition as well as an increase
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in certain enzymatic activities and a decrease in other enzymatic activities catalyzing transformations of the same substrates are the necessary conditions for switching on various interchangeable (shunt) metabolic mechanisms under adverse environmental conditions. Eventually, this provides the stability of metabolism [17–20]. The development of such a nonspecific set of biological changes triggered by Furolan is determined by the potential of a particular genotype and realized by various molecular mechanisms stimulating protein synthesis. The most expedient of these mechanisms are the change in the degree of mRNA polyadenylation and the corresponding alteration in mRNA stability and translational activity. REFERENCES 1. Kul’nevich, V.G., Kalashnikov, V.G., Kosulina, T.P., Nen’ko, N.I., and Smolyakov, V.P., Novye napravleniya v khimii tsiklicheskikh atsetalei (New Directions in Chemistry of Cyclic Acetals), Ufa: Reaktiv, Nova Science Publ. Inc., 2002. 2. Kefeli, V.I., Fiziologicheskie osnovy konstruirovaniya gabitusa rastenii (Physiological Principles of Building the Plant Habitus), Zhuchenko, A.A., Ed., Moscow: Nauka, 1994. 3. Tarchevskii, I.A., Metabolizm rastenii pri stresse (Metabolism of Plants in Stress), Kazan: Fen, 2001. 4. Reymond, Ph. and Farmer, E.E., Curr. Opin. Plant Biol., 1998, vol. 1, no. 5, pp. 404–411. 5. Plotnikov, V.K., Usp. Sovrem. Biol., 1992, vol. 112, no. 2, pp. 186–199.
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