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UDC 581.134.1 https://doi.org/ 10.15407/biotech9.05.064
ICP-MS ANALYSIS OF BREAD WHEAT CARRYING THE GPC-B1 GENE OF Triticum turgidum ssp. dicoccoides
S. Yu. Pokhylko1, 2 institute of Cell Biology and Genetic Engineering
V. V. Schwartau3 of the National Academy of Sciences of Ukraine, Kyiv
L. M. Mykhalska3 2National Technical University of Ukraine "Ihor Sikorsky
O. M. Dugan2 Kyiv Polytechnic Institute", Kyiv
B. V. Morgun1, 3 3Institute of Plant Physiology and Genetics
of the National Academy of Sciences of Ukraine, Kyiv
E-mail: molgen@icbge.org.ua
Received 27.07.2016
The aim of the research was to analyze effects of the gene Gpc-B1, introgressed from wild spelt Triticum turgidum ssp. dicoccoides, on the presence of biologically important elements in bread winter wheat hybrid families of generations F4 and F5. The accumulation of metals in ripe and unripe grains was measured on a mass spectrometer with inductively coupled plasma ICP-MS Agilent 7700x. It was found that the expressing gene significantly increased the content of trace elements Fe, Mn, Zn and Cu in ripe wheat kernels on average by 50-70%, while the increase of secondary nutrients or mesoelements Mg, Ca defined by an average of 20-40%. The enrichment of minerals, confirmed during grain development and ripening provides not only biofortification for the future harvest, but also potentially enhances the resistance of plants to diseases and formation of seedlings with more efficient use of nitrogen.
Key words: Triticum aestivum, biofortification, bread wheat, Gpc-B1.
The content of trace elements within ISO 3768-2010 in wheat grains is important for nutrition and getting a seed culture with high sowing qualities. There are low levels of Zn, Fe and Mn in soils and crops in all the countries, but it is particularly dangerous in regions of growing crops. Those minerals are the basis of human nutrition. The problem of lack of biologically important trace elements such as Fe and Zn is relevant to more than 2 billion people around the world. Pregnant women and children less than 5 years old suffer shortages most [1-4].
In European countries, grains, especially wheat, provide about 30% of daily calorie intake while in the Central Asia go to an average of 50% [1, 4, 5]. The wheat does not always contain sufficient micronutrients to ensure adequate human diet [6].
Micronutrient content in wheat genetically determined and depends on environmental factors. The level of absorption of trace elements in generative organs of wheat is low. Thus, it is a promising trend of genetic improvement of crops. One of the areas of biofortification of micronutrients is
the transfer of genes from wild relatives, including, in particular, as spelt and wild wheat [7, 8]. Gene Gpc (grain protein content) within the whole chromosome 6B was transferred from tetraploid hulled wild emmer Triticum turgidum L. subsp. dicoccoides (Korn. ex Asch. & Graebn.) Thell. in hard tetraploid wheat cultivar Langdon (Triticum turgidum L. var. durum) [7]. The gene is responsible for accelerated aging of leaves and causes the effect of increasing the content of important trace elements in wheat, including Zn, Mn and Fe. The effect locus was detected in a significant increase compared to the starting line in the grain content of zinc (60 to 47.5 mg/kg), iron (44.2 to 35.9 mg/kg), manganese (53.9 to 40.9 mg/kg) and protein (14.4 to 10.8%) [8-10].
Well known that iron, zinc and manganese are some important elements to ensure the redox homeostasis of living organisms. Iron is available to plants in the forms Fe+2 and Fe+3. This element plays an important role in metabolism, is part of enzymes involved in chlorophyll synthesis,
a component Citric acid cycle. The lack of iron during plant growing causes intercostal chlorosis and consequently disrupts the synthesis of chlorophyll and photosynthesis activity [11]. Iron is an essential element in the human body and is a part of many substrates and enzymes responsible for transporting oxygen to cells, functioning respiratory chain mitochondrial redox reactions, the functioning of nervous and immune systems. About 60% of iron in the body is in hemoglobin. The lack of this trace element determines, first of all, the development of hypochromic anemia. The recommended daily dose of iron for adults is 10-15 mg [12-14].
Plants often use zinc as the divalent cation Zn+2. It is one of the most important trace elements necessary for the regulation of carbohydrate and a number of enzymes involved in the growth process. It is a cofactor of more than 80 enzymes and acts as a structural component of many proteins [11]. The role of zinc in human life is due mainly to the fact that it is a member of more than 40 important enzymes that catalyze the hydrolysis of peptides, proteins, certain esters and aldehydes. Zinc is involved in carbohydrate metabolism, located in insulin. The presence of zinc only let vitamin A function. This ion required for bone formation. Lack of zinc in the body has pleiotropic effects on mRNA expression of hundreds of genes, altering the biosynthesis of many proteins and zinc transcription factors. The average daily recommended amount of zinc for adults is 12 mg [2, 15, 16].
Plants absorb manganese in the form of divalent cation Mn+2. It performs a structural role in the chloroplast membrane system [17]. The element deficiency leads to intercostal chlorosis of young and old plant tissues [11]. Manganese is a part of the active site of many enzymes involved in blood clotting, regulates conversion of molecular oxygen. Also manganese is involved in the synthesis of vitamins B and affects the synthesis of hemoglobin. The lack of manganese leads to disruption of carbohydrate metabolism, delays the growth of hair and nails, causes dermatitis, abrupt formation of cartilage, osteoporosis. The daily dose of manganese for adults is 2.0-5.0 mg [17].
Given the biological importance of iron, zinc and manganese, the aim of our study was to analyze the wheat hybrid families of generations F4 and F5 carrying the gene Gpc-B1 from Triticum turgidum
ssp. dicoccoides within the substituted chromosome 6B for the content of micro- and macronutrient in grain.
Materials and Methods
To investigate the content of micro- and macroelements the hybrid wheat families of generations F4 and F5 marked as 18.2, 50.5, 51.5, 94.4, 98.3, 104.5, 125.5, 158.5 bearing the native gene Gpc-B1 of T. turgidum ssp. dicoccoides in Kuyalnyk background (been the second genetic parent) have been selected. The samples of generation F4 were represented by ripe grains while the samples of generation F5 were grains in the milk stage of maturation.
The determination of elements in the grain samples was carried out with a mass spectrometer with inductively coupled plasma ICP-MS Agilent 7700x (Agilent Technologies) together with ICP-MS MassHunter Workstation. The samples (0.400 g) were treated with in nitric acid of ICP-grade in the microwave sample preparation system Milestone Start D. After the cooling, water was added to the samples to the dissolution rate 250x. All solutions were prepared in class 1 water (18 MOm-cm), obtained from the water purification system Scholar-UV Nex Up 1000 (Human Corporation, South Korea). In 2015 Fluka Multielement standard solution 5 for ICP (Switzerland) was used for calibration whereas in 2016 the ICP-MS Complete Standard IV-ICPMS-71A (Inorganic Ventures, USA) was applied which helped identify selenium (Se) too [18, 19]. Settings of the mass spectrometer is given in Table 1.
The selected elements in the argon plasma may produce false peaks. For example, equal by weight CaO56 and Fe56 or ArN or ArO with other isotopes of iron. ArNH and KO may mask Mn, and Ba++ - Zn. All 6 Ca isotopes can interact with O, H and Ar, which leads to inaccuracies in the determination of Cu, Fe, Sc, Se. Determination of selenium prevent most isotopes isobaric overlap Kr or Ge (masses of 74, 76, 78, 80 and 82) or polyatomic interferences mainly on Ar2 masses 76, 78, 80. Although copper ionizes well in argon plasma (90%), the definition isotope 63Cu is complicated with NaAr interaction and types of P, while 65Cu is covered with SO2/ SO2H. Besides, copper isotopes interfere with calcium oxide and titanium. Taking in to account the named obstacles the measurement was performed in helium purge mode, which effectively removes the above matrix and element interference. As an internal standard
Table 1. Setting a mass spectrometer to analyze the Agilent 7700x inorganic elements
Indicator Settings Value
Supply Power generator, W 1550
Setup argon plasma Flow of carrier gas, l/min 15.5
Flow of helium, l/min 0.1
Auto Setup CeO+/Ce+ (%) 1.114
Ce+/Ce+ (%) 1.867
Sensitivity pulses per second to mg/l (CPS/ppb) Li (62700), Y (92920), Tl (87080)
the 1 ppb solution of Sc by Inorganic Ventures, USA was used. The statistical analysis of obtained results was performed with ICP-MS MassHunter Software and MS Excel 2014.
Results and Discussion
As a result of ICP-MS detection of a number of elements contents in mature wheat grains generation F4 8 homozygous hybrid families where parental control served as a cultivar of Kuyalnyk, have the following (Table 2).
Average zinc content among the prototypes of 23.25 mg/kg dry weight, 83% more than the original control (cultivar Kuyalnyk). Maximum accumulation of zinc occurs in the family 94.4 — 33 mg/kg, which is 155% higher than the control cultivar. Minimal accumulation of zinc in the family determined 50.5 — 17 mg/kg of dry matter, but even this level of content significantly exceeded the results of the control options.
The increase in the accumulation of iron in the homozygous hybrid families was slightly higher compared to the levels of accumulation of zinc. The average iron content of test samples is 52.3 mg/kg of dry matter, which, in turn, 61% more than the original line. The maximum iron content was observed in the family 51.5 — 69 mg/kg, minimum — in the family 158.5 — 40 mg/kg.
The average concentration of manganese prototypes of 50.7 mg/kg of dry matter, which is 55% more than the original line. The maximum manganese content was observed in the family 18.2 — 65 mg/kg, minimum — in the family of 158.5 index of 45 mg/kg.
Given the biological importance of other divalent cations detected and copper content as well as magnesium and calcium. This essential elements, high content which determines crop seed quality and nutritional
value of wheat flour. Copper content in grain increased in all variants, but did not reach the size limit accumulation of 10.0 mg/kg, which is regulated by law in accordance with ISO 3768-2010. It was also noted the high content of magnesium accumulation in grains and calcium — in most lines. The average concentration of magnesium, compared with the original line was over 46%, calcium — 24%, copper — 64%.
As a result of ICP-MS analysis of the content of Zn, Fe, Mn, Cu, Mg and Ca in all research family is the high content of micronutrients compared to the cultivar of Kuyalnyk. This finding confirms previous studies [9, 10, 19] that Gpc-B1 gene of Triticum turgidum ssp. dicoccoides positive effect on increasing micronutrient content in its carrier.
In 2016 was analyzed hybrid generation families F5. For research took grain maturation phase of the milk. Served as a control parental line (cultivar Kuyalnyk). The task of the research was to determine the degree of accumulation of micro- and macroelement number of hybrid gene carriers family — Gpc-B1 of Triticum turgidum ssp. dicoccoides the initial phase of ripening grain. Analysis was performed on 6 hybrid families that they investigated in 2015. The data given in Table 3.
The content of zinc in hybrid families an average of 27.6 mg/kg of dry matter, which is 28% more than in the starting line. The maximum level was observed in the family 98.3 — 33 mg/kg.
The average iron content in samples of 2016 amounted to 28.7 mg/kg of dry matter, while the beans control — 27.0 mg/kg (8% difference). The maximum iron content was observed in the family 51.5 — 32.0 mg/kg, the minimum content 104.5 — 27.0 mg/kg. A small difference can be explained by the
Table 2. The content of micro and macroelements in physiologically mature grains of wheat crop 2015 (mg/kg dry matter)
Variant Microelement Macroelement
Zn Fe Mn Cu Mg Ca
Control (cultivar Kuyalnyk) 13a 33a 33a 2.80a 911a 372a
18.2 25c 58e 65d 5.87c 1723d 560d
50.5 17b 46c 47b 4.45b 1257c 399a
51.5 20b 69f 49b 4.09b 1329c 444b
94.4 33d 48c 49b 4.36b 1268c 465c
98.3 21c 57d 52c 4.22b 1351c 403b
104.5 19b 53d 52c 4.37b 1279c 494c
125.5 29d 48c 48b 4.76b 1377c 492c
158.5 22c 40b 45b 4.59b 1023b 434b
Note: the identical letters mark variations that do not differ on the 0.05 significance level.
Table 3. The content of wheat grain micro- and macroelements in the initial stages of ripening at harvest in 2016 (mg/kg dry matter)
Variant Microelement Macroelement
Zn Fe Mn Cu Se Mg
Control (cultivar Kuyalnyk) 21a 27a 49a 4.2a 0.09a 1665a
18.2 24b 29a 63c 5.0b 0.41c 1682a
51.5 27c 32c 60b 5.0b 0.23b 1631a
94.4 26c 28a 59b 5.2b 0.03a 1681a
98.3 33d 29b 63c 6.0c 0.14a 1853b
104.5 24a 27a 65c 5.8b 0.11a 1648a
125.5 31d 30c 77d 7.2d 0.26b 1960c
fact that the grain is not yet mature and the accumulation of iron in the plant continued until fully ripe grain.
The average concentration of manganese incurred samples was 64.6 mg/kg, which is 31% more than in the starting line Kuyalnyk grade. The maximum level was 125.5 in the family — 77 mg/kg and the minimum — in the family 94.4 — 59 mg/kg of dry matter.
Analyzing the performance of content generation F4 and F5 on the content of Zn, Mn, Fe, one can conclude that the gene
Gpc-B1 of Triticum turgidum ssp. dicoccoides significantly increases the content of trace elements in samples. This effect was observed even in the early stages of ripening grain. Also, we have determined the content of Cu and Mg in hybrid families.
Mean values of copper in hybrid families by 35% higher than the content in the starting line, whereas the magnesium content — 5%. Elevated levels of elements at this early stage indicates that the gene Gpc-B1 of Triticum turgidum ssp. dicoccoides multifunctional and
not only increases the amount of Fe, Mn, Zn, but Cu, Mg.
One of the important micronutrients for wheat is also selenium. It is a component of over 30 vital biologically active substances of the human body as part of the active centers of antioxidant enzymes involved in the metabolism of nucleic acids, lipids, hormones. Among plant products the main source of selenium in Ukraine is wheat flour, so control of the content of selenium is essential [20, 21].
The average content of selenium in experimental families of 0.207 mg/kg of dry matter, which is 117% more than the father. The maximum level of selenium — family 18.2 — 0.41 mg/kg (4.5 times more than control) in the dry matter.
According to the research in 2015 and 2016 we found that the presence of the gene Gpc-B1 of Triticum turgidum ssp. dicoccoides bread
winter wheat growth leads to accumulation of important biologically significant nutrition elements — iron, zinc, manganese, copper, selenium, and magnesium (which is correlated with observations of other researchers on a number of cereals [5, 8, 19]). Given the importance of iron, zinc, manganese, copper and selenium in redox homeostasis, we can provide high quality seeds sown grain containing this gene, as well as increased resistance of plants to pathogens destructive diseases — Fusarium spp., etc. Enriching copper ions promote the formation of stairs cultures with high levels of nitrogen use efficiency.
Thus, the presence Gpc-B1 locus of Triticum turgidum ssp. dicoccoides and its functioning is important for a bread winter wheat biofortification and further breeding work.
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ICP-MS АНАЛ1З М'ЯКО1 ПШЕНИЦ1
З ГЕНОМ GPC-B1 В1Д Triticum turgidum ssp. dicoccoides
С. Ю. Похилько1' 2, В. В. Швартау3, Л. М. Михальська3, О. М. Дуган2, Б. В. Моргун1' 3
Институт ^i™HHOï бмлоги та генетично1 шженери НАН Украши, Кшв 2Нащональний техшчний ушверситет Украши «Кшвський полггехшчний шститут iMern 1горя Сшорського», Кшв 31нститут фiзiологiï рослин i генетики НАН Укра1ни, Кшв
E-mail: molgen@icbge.org.ua
Метою роботи був аналiз впливу гена Gpc-B1' штродукованого в^ дикоï полби Triticum turgidum ssp. dicoccoides, на бмлопчно важливi елементи живлення пбридних имей пшенищ m^^ï озимоï поколшь F4 та F5. Накопичення металiв у стиглих та недозр^их зершвках вимiрювали на мас-спектрометрi з iндуктивно зв'язаною плазмою ICP-MS Agilent 7700x. Встановлено, що експресiя цього гена ^тотно пiдвищуe вмiст мiкроелементiв Fe, Mn, Zn i Cu у стиглих зершвках пшенищ m^^ï — у середньому на 50-70%, тимчасом як мезоелеменив Mg, Ca — у середньому на 20-40%. Збагачення мшералами, що шдтверджено шд час розвитку та наливу зерна, не лише забезпечуе б^фортифшащю майбутнього врожаю, але й сприяе пiдвищенню резистентностi рослин до хвороб, формуванню сходiв iз бiльш ефективним використанням азоту.
Ключовi слова: Triticum aestivum, бмфортифь кацiя, м'яка пшениця, Gpc-B1.
ICP-MS АНАЛИЗ МЯГКОЙ ПШЕНИЦЫ С ГЕНОМ GPC-B1 ОТ Triticum turgidum ssp. dicoccoides
С. Ю. Похилько1, 2, В. В. Швартау3, Л. Н. Михальская3, А. М. Дуган2, Б. В. Моргун1, 3
1Институт клеточной биологии и генетической инженерии НАН Украины, Киев 2Национальный технический университет
Украины «Киевский политехнический институт имени Игоря Сикорского», Киев 3Институт физиологии растений и генетики НАН Украины, Киев
E-mail: molgen@icbge.org.ua
Целью работы был анализ влияния гена Gpc-B1, интродуцированного от дикой полбы Triticum turgidum ssp. dicoccoides, на биологически важные элементы питания гибридных семей пшеницы мягкой озимой поколений F4 и F5. Накопление металлов в зрелых и созревающих зерновках измеряли на масс-спектрометре с индуктивно связанной плазмой ICP-MS Agilent 7700x. Установлено, что экспрессия этого гена значительно повышает содержание микроэлементов Fe, Mn, Zn и Cu в зрелых зерновках пшеницы мягкой — в среднем на 50-70%, в то время как мезоэлементов Mg, Ca — в среднем на 20-40%. Обогащение минералами, подтвержденное при развитии и наливе зерна, не только обеспечивает биофортификацию будущего урожая, но и способствует повышению резистентности растений к болезням, формированию всходов с более эффективным использованием азота.
Ключевые слова: Triticum aestivum, биофортификация, мягкая пшеница, Gpc-B1.
