Jerusalem Artichoke: Quality Response to Potassium Fertilization and Irrigation in Poland

: The aim of this study was to determine the e ﬀ ects of soil potassium fertilization (150, 250 and 350 kg K 2 O ha − 1 ) and irrigation on the tuber quality (content of ´ α -tocopherol, β -carotene, essential and endogenous amino acids) of three Jerusalem artichoke ( Helianthus tuberosus L.) cultivars (Topstar, Violette de Rennes, Waldspindel). Jerusalem artichokes were grown during a ﬁeld experiment in the Agricultural Experiment Station in Tomaszkowo (53 ◦ 42 (cid:48) N, 20 ◦ 26 (cid:48) E, north-eastern Poland). The content of ´ α -tocopherol and β -carotene was determined at 1.60–2.65 and 0.75–1.00 mg kg − 1 DM, respectively, in all Jerusalem artichoke cultivars produced in north-eastern Poland. High rates of potassium fertilizer (250 and 350 kg K ha − 1 ) increased the content of ´ α -tocopherol in tubers by 47% and 66% on average, respectively. The stimulatory e ﬀ ects of high potassium rates on the content of ´ α -tocopherol (2.5-fold increase) were observed only in response to irrigation. High rates of potassium fertilizer induced a particularly high increase (3.2-fold) in ´ α -tocopherol concentrations in Jerusalem artichokes cv. Waldspindel. Irrigation increased ´ α -tocopherol levels (by 40%) and decreased the concentrations of β -carotene (by 25%) and most essential and endogenous amino acids (isoleucine, leucine, lysine, phenylalanine, valine, alanine, glycine, histidine, serine, threonine). The Topstar cultivar accumulated the highest quantities of essential and endogenous amino acids. Leucine, methionine + cysteine were the limiting amino acids in Jerusalem artichoke tubers. The analyzed tubers were characterized by very high nutritional quality of dietary protein (Essential Amino-Acid Index, 66–78).


Introduction
Jerusalem artichoke (Helianthus tuberosus L., Asteraceae) originated in North America where it was cultivated by the native population [1,2]. The species is presently grown in Europe and Asia as a valuable source of lignocellulosic biomass for the energy sector [3][4][5][6][7][8][9]. Jerusalem artichoke is a crop species that most effectively converts solar energy to biomass in both quantitative and qualitative terms [10][11][12][13][14]. Processed biomass is a valuable resource for the production of biofuels [1,3,4,15,16]. Immature aerial parts of H. tuberosus are used in biogas production, whereas straw is processed into solid fuels (pellets, briquettes), and tubers are a high-yielding resource in the production of bioethanol or substrate/cosubstrate in the methane fermentation process [16][17][18]. In the temperate climate of Poland, tuber and straw yields can reach 14-30 and 20-50 Mg ha −1 dry matter (DM), respectively [13]. The amount of energy accumulated in the harvested biomass (tubers, straw) ranges from 142-281 [4] to 348 GJ ha −1 [19]. However, high levels of genetic/phenotypic variation in H. tuberosus populations lead to considerable differences in yield, even in relatively similar agricultural production systems in Europe [13,20]. Jerusalem artichoke has low agronomic requirements [1,16], it is resistant to a wide range of biotic and abiotic stressors [1,[21][22][23], and prevents soil erosion [17]. The species easily adapts to diverse environments [8], therefore it can be effectively grown for energy production on marginal land [4]. Jerusalem artichoke is also a natural source of: (i) inulin, (ii) oligofructose, and (iii) fructose [24][25][26], compounds with nutritional and functional attributes (food and feed) that are particularly beneficial to individuals with type 2 diabetes, obesity and cardiovascular disorders [25,27,28]. Tubers are abundant in protein, polyphenols and vitamins [26,29]. These qualities make Jerusalem artichoke biomass particularly suitable for biorefining and the development of the bioeconomy [6]. In biorefineries, organic chemical compounds and lignocellulosic materials are recovered, processed and used in the production of biofuels, bioproducts and bioenergy. Jerusalem artichoke biomass is ideally suited for these industrial applications [23].
Jerusalem artichoke tubers can survive (without loss) in frozen soils for several months because they are abundant in inulin. Overwintering success is compromised only in areas where tubers become dehydrated [21]. The species has minimal soil and fertilizer requirements [21,28,30]. It is susceptible to numerous fungal pathogens (Sclerotinia sclerotiorum (Lib.) de Bary, Fusarium acuminatum Ellis & Everh, Botrytis cinerea Pers.) which colonize mainly tubers. Pests of economic importance do not exert pressure on Jerusalem artichoke plantations [23].
The biological value of Jerusalem artichoke tubers is determined by the genetic characteristics of cultivars and the applied production technology [28,[30][31][32][33][34]. Fertilization, in particular potassium fertilization, is an agricultural treatment that exerts the greatest impact on the biological value of tubers. Potassium enhances plant growth, development and tuber yields [35], and it plays an important role in the transport of photosynthesis products from leaves and their accumulation in tubers [36,37]. Agricultural crops are increasingly often irrigated due to a higher risk of drought, including in the temperate climate [38][39][40]. Denorpy [17], Monti et al. [24] and Gao et al. [34] demonstrated that H. tuberosus is sensitive to water stress. Jerusalem artichoke thrives in regions with high levels of precipitation, and irrigation exerts particularly beneficial effects during tuber formation [34]. The aim of this study was to determine the effects of soil potassium fertilization and irrigation on the tuber quality (measured as content ofά-tocopherol, β-carotene, amino acids) of three Jerusalem artichoke cultivars representing different maturity groups.

Field Experiment
Jerusalem artichoke (Helianthus tuberosus L.) was grown in a field experiment in the Agricultural Experiment Station in Tomaszkowo (53 • 42 N, 20 • 26 E, NE Poland) in 2018. The experiment had a three-factorial split-split-plot design with three replications. The experimental variables were: (i) cultivar: Topstar (an early edible cultivar with yellow-brown tubers), Violette de Rennes (a mid-late edible cultivar with red tubers), Waldspindel (a mid-late cultivar with red tubers, used in herbal and distilling industries); (ii) soil potassium fertilization-potassium sulfate (kg K 2 O ha −1 ): 150, 250, 350; (iii) irrigation: with and without irrigation.
Soil moisture content was controlled from the beginning of tuber formation, during the growth of aerial plant parts, until leaf ageing and the translocation of sugars to tubers (from mid-June to mid-October). The optimal soil moisture content was set at 14.3-16.5%, i.e., 65-75% of field water capacity at a depth of 30 cm. Measurements were conducted twice a week. The crops were irrigated every 5-7 days at 20 dm 3 m −2 when field water capacity decreased below 60% (≤13.2% soil moisture content). Irrigation treatments for Jerusalem artichoke were developed based on the irrigation regime for late potato cultivars (Solanum tuberosum L.) and field water capacity for different soil types [40]. Soil moisture content was measured with the SM 150-KIT probe (Geomor-Technik sp. z o. o., Szczecin, Poland). Plots were irrigated 11 times during the growing season of H. tuberosus (13 and 19 June; 3 and 9 July; 10 and 23 August; 3, 11, 20 and 28 September; 6 October) in the total amount of 220 mm of water.
The experiment was established on Haplic Luvisol loamy sand [41]. Each experimental plot had an area of 2.7 m 2 . Oat (Avena sativa L.) was the preceding crop. Composite soil samples were collected from each plot to a depth of 20 cm to determine the chemical properties of soil. Soil pH was determined at 5.4 with a digital pH meter, and soil nutrient levels were determined at 74 mg P kg −1 (Egner-Riehm method), 145 mg K kg −1 (Egner-Riehm method) and 69 mg Mg kg −1 (AAS) [42]. Tubers were hilled once after planting. Jerusalem artichoke was harvested at the beginning of November.

Determination of Amino Acid Content and the Nutritional Value of Protein
The content of amino acids, excluding tryptophan, was determined in dried tuber samples in the AAA-400 amino acid analyzer (INGOS s.r.o, Prague, Czech Republic). The samples were hydrolyzed in 6 M HCl for 24 h at a temperature of 110 • C. The hydrolysate was cooled, filtered, rinsed and evaporated in a water bath, and dry residue was dissolved in a buffer with a pH of 2.2. The obtained specimens were analyzed in the ninhydrin test. The applied buffers had a pH of 2.6, 3.0, 4.25 and 7.9. Ninhydrin solution was buffered at pH 5.5. The column with a length of 370 mm was packed with ion-exchange resin (Ostion ANB, INGOS s.r.o.). Column temperature was 58-74 • C, and reactor temperature was 120 • C. The concentrations of sulfur-containing amino acids (methionine and cysteine) were determined by hydrolysis with a mixture of formic acid and hydrogen peroxide (9:1) at a temperature of 110 • C for 23 h. Cooled samples were handled in accordance with the acid hydrolysis protocol. The applied buffers had a pH of 2.6 and 3.0. Column temperature was 60 • C, and reactor temperature was 120 • C. Tryptophan content was determined based on Polish Standard PN-77/R-64820 [43]. The applied analytical method involves the quantification of synthetic methionine, but not the hydroxy analogue of methionine.
The nutritional value of protein was determined with the use of the limiting amino acid index [47] (Equation (1)) and the essential amino acid index (EAAI) [48]: where CS -chemical score, a b -content of essential amino acid and a w -content of essential amino acid in the reference protein.

Statistical Analysis
Data were processed statistically by one-way analysis of variance (ANOVA) in the Statistica 13.3 program [49]. Multiple comparison Tukey's HSD (honestly significant difference) statistical test was applied to assess significant differences (p < 0.05) between means. The results of the F-test for fixed effects in ANOVA are presented in Table 1.

Weather Conditions
The growing season of 2018 lasted for 201 days. Jerusalem artichokes were harvested in the first week of November. Weather conditions during the experimental period are presented in Table 2. During the growing season, mean monthly air temperatures did not differ considerably from the long-term average of 1981-2010. Rapid growth of aerial plant parts and tuber setting began in mid-June. Total precipitation during the growing season reached 418.8 mm, and it was 7% lower than the long term average (450.1 mm). Rainfall was unevenly distributed, with dry spells in May and September. Precipitation levels were lowest in May and September, 57% and 64% lower than the long-term average, respectively, and irrigation was required (3, 11, 20 and 28 September). As described in the Methods section, the optimal moisture content of soil was determined based on the irrigation system for late-maturing potato varieties. A comparison of the water requirements of plants with precipitation levels shows that rainfall was 27% lower than the optimal water supply also in August. Only in July precipitation exceeded the long-term average by 90% and the water requirements of plants by 45%. Despite the above, measurements of the soil moisture status revealed the need for irrigation since the field water capacity dropped below 60%, i.e., to ≤13.2%.
In the current study, an increase in the potassium fertilizer rate led to a significant increase in theά-tocopherol content of Jerusalem artichoke tubers (Table 3). This correlation was particularly pronounced in cv. Waldspindel whereά-tocopherol levels increased 3.2-fold when the potassium rate was increased from 150 to 350 kg K 2 O ha −1 (Figure 1). Irrigation increasedά-tocopherol concentrations by 41% on average in the studied Jerusalem artichoke cultivars (Table 3). Potassium fertilizer enhanced α-tocopherol levels only in irrigated plots. In non-irrigated plots, potassium fertilization was not correlated withά-tocopherol concentrations in H. tuberosus tubers (Figure 2). α-Tocopherol is the most biologically active form of tocopherol, and its proportion in total tocopherols is an important consideration [52]. Potassium rate was the only experimental variable which significantly differentiated the proportion of α-tocopherol in total tocopherols ( Table 1). The highest percentage of α-tocopherol in total tocopherols (79.6% on average) was noted in tubers supplied with potassium at 350 kg ha −1 , regardless of cultivar or irrigation (Table 3).
β-Carotene is a vitamin A precursor which is essential for the maintenance of normal vision, healthy skin and immunity [58]. The recommended daily allowance of vitamin A is 4.8 mg of β-carotene [59]. Foods of animal origin and carotene-containing plants are the main sources of vitamin A in the human diet [52,60,61]. In this study, the β-carotene content of H. tuberosus tubers ranged from 750 to 1000 µg kg −1 DM (Table 3), and it was 1.8-to 2.5-fold higher than in potato tubers (400 µg kg −1 DM), and 3.7-to 5-fold higher than in sugar beetroots (200 µg kg −1 DM) [62]. In the present study cultivar, potassium rate and irrigation did not affect β-carotene synthesis in Jerusalem artichoke tubers (Table 1). In a study by Wierzbicka and Hallmann [61], agronomic factors also exerted a weak influence on β-carotene synthesis in potato tubers.  Vitamin A activity is determined in retinol equivalents (RE) [46,62]. Raju et al. [46] analyzed the activity of provitamin A in 30 species of leafy vegetables and reported the highest value in broccoli (641 RE) and the lowest value in sorrel (12 RE). In the present experiment, vitamin A activity in Jerusalem artichokes was determined in the range of 125-167 RE, and it was higher in non-irrigated tubers (Table 3).

Amino acid Content and Nutritional Value of Protein
Jerusalem artichoke contains highly nutritional protein with a balanced amino acid profile [63]. In this study, amino acid concentrations differed across the examined cultivars ( Table 1).
The highest content of most essential (isoleucine, leucine, phenylalanine, thyrosine and valine) and endogenous amino acids (alanine, glycine, threonine) was noted in cv. Topstar. Jerusalem artichoke cv. Waldspindel was most abundant in thyrosine and arginine, whereas the tubers of cv. Violette de Rennes were characterized by the highest concentrations of aspartic acid and glutamic acid (Tables 4 and 5).
The content of lysine, methionine, cystine, tryptophan, histidine, proline and serine was not differentiated by the genetic factor (Table 1). An increase in the potassium fertilizer rate to 250 kg K 2 O ha −1 decreased the content of glutamic acid (by 1.22 g 100 g −1 of protein) in Jerusalem artichoke tubers (Table 5). This correlation was particularly visible in cvs. Violette de Rennes and Topstar where glutamic acid levels decreased by 2.57 g 100 g −1 of protein on average in response to higher potassium rates (Figure 3). The phenylalanine content of the examined cultivars was also influenced by potassium fertilization (Cv. × K interaction) ( Table 1). Phenylalanine levels were highest in cv. Topstar supplied with the lowest potassium rate (150 kg K 2 O ha −1 ). In the tubers of cv. Waldspindel, similar concentrations of phenylalanine were noted only in response to the highest potassium rate (350 kg K 2 O ha −1 ) (Figure 4).    In the work of Eppendorfer [64], potassium deficiency induced the greatest decrease in the concentration of proline, followed by glutamic acid, but it increased histidine and aspartic acid levels. These findings were not corroborated by the present study where the synthesis of endogenous amino acids was not significantly affected by higher potassium rates ( Table 5). The content of aspartic acid in Jerusalem artichoke tubers was a cultivar-dependent trait ( Table 1). The highest accumulation of aspartic acid was noted in cv. Violette de Rennes (Table 5). According to Eppendorfer [64], as cited by Pęksa et al. [65], the content of essential amino acids is inversely correlated with fertilizer rates. In this experiment, the applied rates of potassium fertilizer were not correlated with the synthesis of essential amino acids (isoleucine, leucine, lysine, methionine, cystine, phenylalanine, thyrosine, tryptophan, valine).
Irrigation decreased the content of most essential (isoleucine, leucine, lysine, phenylalanine, valine) and endogenous amino acids (alanine, glycine, histidine, serine, threonine) (Tables 4 and 5). A particularly high decline (25%) in valine concentration was noted in irrigated tubers cv. Topstar ( Figure 5). Irrigation enhanced the concentrations of only selected endogenous amino acids (arginine, glutamic acid, proline) (Tables 4 and 5). In irrigated plots, an above-average increase was noted in arginine levels in tubers cv. Waldspindel and in glutamic acid and proline concentrations in cv. Violette de Rennes (Figures 6-8).    The essential amino acid index and the limiting amino acid index support evaluations of the nutritional quality of protein relative to chicken egg protein as the reference. New reference proteins which contain smaller amounts of amino acids (even twice smaller for methionine + cysteine, threonine, tryptophan, phenylalanine + thyrosine) and correspond to human nutritional requirements have been introduced in 2007 [66]. The nutritional value of the studied protein preparations was evaluated based on their chemical scores (SC). The chemical scores of cvs. Topstar and Waldspindel exceeded the reference value only for phenylalanine + thyrosine (by 29.8% and 18.2%, respectively), and leucine was the limiting amino acid (52.7% and 45.0%, respectively). In a study by Mitrus et al. [67], leucine was also the first limiting amino acid in S. tuberosum (36.5%). In Jerusalem artichokes cv. Violette de Rennes, the content of all amino acids was below the reference value, from 1.6% (phenylalanine + thyrosine) to 21.8-59.2% (the remaining amino acids), and methionine + cysteine were the limiting amino acids (40.8%) ( Table 6). Methionine + cysteine were also identified as the limiting amino acids in Jerusalem artichoke tubers analyzed by Danilcenko et al. [68] and Pęksa et al. [69]. In the work of Ciborowska and Rudnicka [70], the nutritional value of potatoes was limited by methionine + cysteine. According to Zhu et al. [71] and Pęksa et al. [65,69], the limiting amino acids in potato and Jerusalem artichoke tubers are determined mainly by cultivar and geographic location. Jerusalem artichokes are abundant in protein of high biological value. The species contains all essential amino acids in optimal proportions. Cieślik and Filipiak-Florkiewicz [63] reported higher levels of methionine in Jerusalem artichoke than in potato tubers. Contrary results were noted in this study, in particular in cv. Violette de Rennes. The nutritional value of protein evaluated based on the EAAI ranged from 66% (cv. Violette de Rennes) to 71-78% (cvs. Topstar and Waldspindel) ( Table 6). Oser [48] and Sawicka [23] determined the EAAI of potatoes at 56-68%. The EAAI of other tuber crops of economic importance ranges from 54% (cassava, Manihot esculenta Crantz) to 82% (sweet potatoes, Ipomoea batatas L./Poir.) [48]. The EAAI of Jerusalem artichoke tubers exceeds the mean values for S. tuberosum and M. esculenta. The chemical score calculated for different potassium fertilization rates exceeded the reference value for phenylalanine + thyrosine (by 12% at 250 kg K 2 O ha −1 , by 14% at 150 kg K 2 O ha −1 , and by 20% for 350 kg K 2 O ha −1 ) ( Table 7). The limiting amino acid was leucine at 150 kg K 2 O ha −1 (47%), and methionine + cysteine at 250 and 350 kg K 2 O ha −1 (45% and 49%, respectively). The nutritional value of protein expressed by the EAAI relative to the reference protein ranged from 70 to 74% for the applied rates of potassium fertilizer (Table 7). The CS of phenylalanine + thyrosine exceeded the reference value by 10% in irrigated plots and by 20% in the absence of irrigation (Table 8). The limiting amino acid was leucine regardless of the irrigation regime (44-52%). The nutritional value of protein expressed by the EAAI relative to the reference protein ranged from 67 to 76% in irrigated and non-irrigated tubers (Table 8).

Conclusions
The present study confirmed that the genetic factor (cultivar) strongly affects the biological value of Jerusalem artichoke tubers. Topstar cultivar was most abundant in essential (isoleucine, leucine, phenylalanine, thyrosine, valine) and endogenous amino acids (alanine, glycine, threonine). High rates of potassium fertilizer (250 and 350 kg K 2 O ha −1 ) increased the absolute content ofά-tocopherol and its proportion in total tocopherols, and decreased the content of glutamic acid in tubers. Irrigation increasedά-tocopherol levels and decreased the concentrations of β-carotene and most essential and endogenous amino acids. The limiting amino acids in Jerusalem artichoke tubers were leucine (cv. Topstar and Waldspindel) and methionine + cysteine (cv. Violette de Rennes). Leucine was the limiting amino acid in tubers supplied with low potassium rates (150 kg K2O ha −1 ), whereas in plots with moderate and high potassium rates (250 and 350 kg K 2 O ha −1 , respectively), the limiting amino acids were methionine + cysteine. Jerusalem artichoke tubers were characterized by very high nutritional value of protein, expressed by the EAAI.
Author Contributions: Conceptualization, methodology, B.B., formal analysis, writing-original draft preparation, B.B. and K.J. All authors have read and agreed to the published version of the manuscript.
Funding: Project financially co-supported by Minister of Science and Higher Education in the range of the program entitled "Regional Initiative of Excellence" for the years 2019-2022, Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN."