
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

72472
10.1038/s41598-024-72472-2
Article
Effect of potassium on the agronomic traits and fruit quality of Goji (Lycium barbarum L.)
Wan Ru
Shi Zhigang shizhigang76@163.com

Li Yuekun linda28772877@163.com

Huang Ting
Cao Youlong
An Wei
Zhang Xiyan
Zhao Jianhua
Qin Ken
Wang Xiao
Yang Libin
https://ror.org/019dkz313 grid.469610.c National Wolfberry Engineering Research Center, Wolfberry Science Research Institute, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan, 750002 China
14 9 2024
14 9 2024
2024
14 2147728 2 2024
9 9 2024
© The Author(s) 2024
2024
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To investgate the effects of potassium (K) application on the agronomic traits and fruit quality of Lycium barbarum L. (Goji), three levels of K fertilizer, namely LK (25 g/plant), CK (50 g/plant), and HK (75 g/plant), were applied to plants in phytotron for observing and measuring relevant indicators. The investigation involved seven agronomic traits: plant height, plant stem diameter, new branch increment, yield of fresh fruits per plant, dry fruit quantity within 50 g, ratio of different grade fruits, and ratio of longitudinal diameter to transverse diameter of Goji fruits. The results showed that K application level had significant effect on ratio of the longitudinal diameter to the transverse diameter of fresh Goji fruits, and that the influence on other agronomic traits was slight. In the meanwhile, the concentrations of amino acids, betaine, polysaccharides and flavonoids of Goji fruits in different levels of K fertilizer were tested. The K treatment increased the content of glutamic acid, and decreased that of flavonoids (P < 0.05), whereas the content of other amino acids, polysaccharides and betaine were unaffected. A total of 132 flavonoid metabolites was identified. Among them, K treatment up-regulated 36 metabolites and down-regulated 30 metabolites (P < 0.05). The results provided a basis for balanced K supply to regulate the agronomic traits and nutrients of Goji fruits.

Keywords

Lycium barbarum L. fruit
Potassium
Flavonoid
Quality
Agronomic traits
Subject terms

Metabolomics
Plant physiology
National Natural Science Foundation32160062 32160062 32160062 32160062 32160062 32160062 Wan Ru Shi Zhigang Li Yuekun Huang Ting Wang Xiao Yang Libin Key R&D plan project of Ningxia Hui Autonomous Region2021BEF0200102 2021BEF0200102 2021BEF0200102 2021BEF0200102 2021BEF0200102 2021BEF0200102 2021BEF0200102 2021BEF0200102 2021BEF0200102 2021BEF0200102 Wan Ru Shi Zhigang Li Yuekun Cao Youlong An Wei Zhang Xiyan Zhao Jianhua Qin Ken Wang Xiao Yang Libin Natural Science Foundation of Ningxia2023AAC03399 2023AAC03399 Wan Ru Shi Zhigang the Special Foundation for Agricultural Breeding of the Ningxia Hui Autonomous Region2018NYYZ0106 2018NYYZ0106 2018NYYZ0106 2018NYYZ0106 2018NYYZ0106 2018NYYZ0106 2018NYYZ0106 2018NYYZ0106 2018NYYZ0106 Wan Ru Shi Zhigang Li Yuekun Huang Ting Cao Youlong An Wei Zhang Xiyan Zhao Jianhua Qin Ken The sixth batch of autonomous region youth science and technology talent promotion project in 2021issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Lycium barbarum L., also known as Goji1, belongs to the genus Lycium in the family Solanaceae. It is one of the major crops in Northwest China, being mainly cultivated in Ningxia, Qinghai, and Tianjin2. Ningqi series belong to L. barbarum are recorded as a medicine3,4 and have high nutritious value5,6. The active constituents are polysaccharides, flavonoids, betaine, vitamins and other nutrients7.

Potassium (K) is an essential nutrient for plant growth and development, and consequently of fertilizers. Its content in the plants is second only to nitrogen (N)8. In addition, K application can also increase the disease resistance of plants9. For example, with the increase of K application in tobacco leaves, the incidence of black shank disease and black rot disease also showed a clear downward trend10. Similarly, the Chrysanthemum morifolium Ramat. in K deficiency will contribute to a significant decline in the physiological resistance of plants. It is prone to leaf blight disease, owing to physiological lack of K, resulting in yield reduction11. This phenomenon also occurs in Goji, causing abscission of leaves, flowers and fruits, and reduced fruit yield12. Potassium is essential for plant growth and function13. A sufficient supply of K improves the contents of amino acids (cystine, methionine, tyrosine, and tryptophan), sugar and acidity in fruits14, mainly because a number of enzymes requiring K + activation, making it essential for normal cell metabolism15. In the northern part of China, even soils with slightly higher K content can suffer from K deficiency in high-yielding and K-loving crops due to factors such as soil drought. Nevertheless, excessive application of K will not only pollute the environment, but also destroy the nutrient balance and lead to the deterioration of crop quality11,16–18. Therefore, the rational application of K fertilizer is crucial for the healthy growth and quality improvement of plants.

The effect of K on the quality of many crops is already explored. The study has shown that spraying the foliar fertilizer potassium dihydrogen phosphate (KH2PO4) on grapes can increase the expression of anthocyanin synthesis genes in fruits19. N and K can change the content of soluble sugars and organic acids in grapes by inducing the expression of the relevant genes in saccharide metabolism, and in turn, regulate anthocyanin synthesis20. For the root of Hedysarum polybotrys Hand.-Mazz., spraying of moderate concentrations of K salts is beneficial to plant growth and isoflavonoid accumulation16. It has demonstrated that with the increase of K application, the content of soluble sugars10, total phenols, flavonoids10,11,17,18, chlorogenic acid11, ascorbic acid17 and lignin18 in plant leaves and flowers increased gradually. The studies indicate that the PAL11,18, PPO and POD18 activities are positively associated with K application, but the content of soluble amino acid is negatively correlated with K application11. It has shown that flavonoid content in both C. morifolium and Labisia Pumila Benth showed an increasing and then decreasing trend with increasing K application. This is because the activities of antioxidant enzymes and nutrients are lower in excessive K content11,17. Liu21 held that K deficiency could significantly reduce the flavonoid content in C. morifolium by 31.4%. Therefore, insufficient or excessive supply of K fertilizer would reduce the activities of metabolic enzymes, the synthesis of phenolics and lignin, and the yield of crops11,17,18.

Lycium barbarum L. is an important economically traditional plant. Potassium is one of the most abundant element in goji fruits22. It was illustrated that soil potassium and AM fungi application promoted L. barbarum growth23. Application of potassium not only improved photosynthesis, including the net photosynthetic rate increased and the intercellular CO2 concentration reduced, but also increased leaf relative water content24. The K + contents of the root in moderate salinized soils were significantly higher than those in mildly or severely salinized soils. Otherwise, the opposite results showed in the stem. Also, the K + contents in leaves and fruits were gradually increased with salinization25. In other words, in severely salinized soils, K + were almost totally absorbed by stem, leaves and fruits. Merely K + contents and extremely high Na + contents in the root results in ion-induced injury of plant cells26. Therefore, increasing K + contents could improve salt resistance ability in plants. The content of flavonoids differs among varieties in response to different genetic backgrounds. It has a wide range of functions in plant tissue colouration and response to adversity27. There are studies on the changes of flavonoid content in leaves and fruits before ripening of Goji fruits28, as well as studies on key genes related to flavonoid synthesis29,30, and studies on the yields and nutrient compositions of Lycium ruthenicum Murray after spraying with KH2PO431. Although there have been some studies on the changing of flavonoid content in Goji fruits during growth and development, the effects of K fertilizer on agronomic traits and quality of Goji fruits are not clear. In this study, the main cultivar "Ningqi 7" was used as the test crop, and we observed the effects of K treatments on the agronomic traits and nutrients of the plants during different harvesting periods. The data, thus provide a theoretical basis for balanced K application to regulate the quality of Goji fruits.

Results

Effects of K fertilization on different agronomic traits

The agronomic traits data was analyzed statistically. The results showed that K treatments have no effects (p > 0.05) on the plant height (Fig. 1A), plant stem diameter (Fig. 1B), new branch increment (Fig. 1C), yield of fresh fruits per plant (Fig. 1D), dry fruit quantity of 50 g (Fig. 1E) and the ratio of different grade fruits (Fig. 1F). As Goji fruit was picked four times (6 June, 25 June, 4 July, 12 July), the agronomic traits at each time were analyzed. The results showed that in three K treatments, the yield of fresh fruits at the second harvest was the highest, and the first harvest was the lowest (Fig. 1G). Moreover, the dry fruit quantity of 50 g at the fourth harvest was more than that at the third harvest, followed by the second harvest at three K levels (Fig. 1H). It indicated that the fruits at the fourth harvest were the smallest, while those at the second harvest were the largest size and highest yield. Owing to larger fruits having higher market prices and greater purchasers, the second-harvest fruits are more popular. The ratio of different-grade fruits was analyzed (Fig. 1I). It clarified that the proportion of first-grade fruits was the highest in both CK (49.17%) and HK (50.75%) treatments.Fig. 1 Agronomic traits of Goji fruit under different K fertilization. (A) plant height; (B) plant stem diameter; (C) new branch increment; (D) yield of fresh fruits per plant; (E) dry fruit quantity of 50 g; (F) ratio of different grade fruits. Agronomic traits of Goji fruit at different harvests: (G) yield of fresh fruits per plant; (H) dry fruit quantity of 50 g; (I) ratio of different grade fruits. Small letters indicate a significant difference (p < 0.05), as analyzed by Duncan’s multiple tests; values without letters mean no significant difference.

The results also showed that K had a significant effect on the ratio of longitudinal and transverse diameters of fresh Goji fruits (p < 0.01) (Fig. 2). The longitudinal diameter and transverse diameter of Goji fruits differ from K treatments (Fig. 2A). They were maximum at HK. The ratio of longitudinal diameters and transverse diameters increased with the increasing K application (LK: 0.93; CK: 1.00; HK: 1.07). At the first and second harvests, the ratio had a significant difference between LK and HK treatments (p < 0.05). The ratio value of HK was higher than that of LK. There were no differences in both third and fourth harvests (p > 0.05). It indicated that the influence of K on the ratio was mainly influenced by the first and second harvests. The fruit shape was significantly different under three levels of K, being flatter at LK, rounder at CK, and longer at HK.Fig. 2 The relationship between longitudinal diameter and transverse diameter under different K fertilization. (A) Longitudinal and transverse diameter of Goji fruits at different levels of K; (B) ratio of longitudinal diameter to transverse diameter of Goji fruits at three K treatments; small letters indicate a significant difference (p < 0.05), as analyzed by Duncan’s multiple tests; values without letters mean no significant difference.

Effect of K on nutrient composition of Goji fruits

The fruits at the second harvest (25 June) had the highest yield (Fig. 1D) and the largest size (Fig. 1E), while the fruits at the fourth harvest (12 July) had the smallest size. To observe the nutrient differences at these two harvest times, the key components (amino acids, betaine, polysaccharides, and total flavonoids) of the fruits were detected.

A total of 17 amino acids were detected (Fig. 3A). Potassium (K) had a significant effect on glutamate between HK and LK (p < 0.05). The glutamate content of HK (1.087 g/kg) was higher than that of LK (0.954 g/kg). It also found that the content of nine amino acids (proline, alanine, valine, leucine, threonine, aspartic acid, methionine, glycine, and tyrosine) had significant effects between the second and fourth harvests (p < 0.05) (Fig. 3B). Among them, the contents of proline, leucine, valine, glycine, and aspartic acid increased over time, while those of threonine, alanine, methionine, and tyrosine decreased. The total content of amino acids on 25 June was lower than that on 12 July in both LK and CK treatments (p < 0.05) (Fig. 3C). However, there was no significant difference in HK (p > 0.05).Fig. 3 Amino acid contents in Goji fruits. (A) Amino acid contents of Goji fruits under different K levels; (B, C) amino acid and total amino acid contents of Goji fruits in different phenology; small letters indicate a significant difference (p < 0.05), as analyzed by Duncan’s multiple tests; values without letters mean no significant difference.

The content of flavonoids negatively correlated with K fertilization (Fig. 4A). It was significantly different from LK and HK (p < 0.05). However, K treatments had no effect on the content of betaine (Fig. 4C) and polysaccharides (p > 0.05) (Fig. 4E). The results also showed that the betaine contents on 12 July (0.7265 g/100 g) were significantly higher than those on 25 June (0.5495 g/100 g) (p < 0.01) (Fig. 4D). However, the content of flavonoids (Fig. 4B) and polysaccharides (Fig. 4F) between the second and fourth harvests was unaffected. It indicated that the content of amino acids and betaine in smaller fruits of "Ningqi 7" was higher than that in larger fruits. Therefore, there isn’t a positive correlation between fruit size and nutritional value.Fig. 4 Flavonoids, betaine and polysaccharides contents in Goji fruits. (A, C, E) The content of flavonoids, betaine and polysaccharides in Goji fruits under different levels of K; (B, D, F) the content of flavonoids, betaine and polysaccharides in Goji fruits in different phenology; small letters indicate a significant difference (p < 0.05), as analyzed by Duncan’s multiple tests; values without letters mean no significant difference.

KEGG pathway analysis of flavonoid metabolites in Lycium barbarum L.

Since K fertilizer had a significant effect on the flavonoid content of Goji (Fig. 4A), flavonoid metabolites were detected. Flavonoids are synthesized through the phenylpropanoid pathway (Fig. 5)9,32. Phenylalanine transformed to p-coumaroyl-CoA and then entered the flavonoid biosynthesis pathway. In this study, 132 flavonoid metabolites, including 42 flavones, 27 flavonols, 2 flavonolignans, 24 flavone C-glycosides, 16 flavanones, 6 isoflavones, 13 anthocyanins, and 2 proanthocyanidins, were identified from Goji fruits. According to the pathway of the Kyoto Encyclopedia of Genes and Genomes (KEGG), there were only 6 flavones, 6 anthocyanins, 6 isoflavones, 9 flavanones, 13 flavonols, and 2 flavonolignans of goji fruits that can be annotated in the pathway (Fig. 5A). By comparing LK with CK, 27 flavonoid metabolites, with 15 being up-regulated and 12 being down-regulated, had changed. By comparing HK to CK, 12 flavonoid metabolites, including 7 up-regulate and 5 down-regulate, had significant differences. There were also 27 metabolites of flavonoid that had significantly changed when comparing LK to HK, with 14 and 13 of them being up-regulated and down-regulated respectively. The enrichment classification results indicated that the impact of K on each group was mainly related to the biosynthesis of flavonoids (Fig. 5B).Fig. 5 Enrichment analysis on Kyoto Encyclopedia of Genes and Genomes (KEGG), which was copyrighted by Kanehisa laboratories32; the p-value represents the degree of enrichment, and the closer the p-value is to 0, the more significant the enrichment is. The size of the point indicates the number of differential metabolites.

PCA analysis of flavonoids metabolites of L. barbarum L. under different K treatments

PCA can indicate that the trend of metabolic group separation between groups and reveal the internal structure between multiple variables. Due to the small distance between the samples within the same group, the similarity between the samples is very high. In the present study, two principal components, PC1 (42.7%) and PC2 (23.6%) were extracted (Fig. 6) and PC1 has the most influence on the variation of the samples. The PCA results showed that LK, HK, and CK were clearly separated, and the repeated samples were compactly gathered.Fig. 6 Analysis of flavonoid metabolites on the basis of principal component analysis.

Analysis of flavonoid content of L. barbarum L. fruit under different K treatments

The trends of flavonoids at different levels of K fertilization were investigated (Fig. 7). Clearly, the total flavone contents decreased with increasing K supply (Fig. 7A), which was 0.18 (g/100 g FW) at LK and 0.105 (g/100 g FW) at HK. There was a significant difference between HK and CK, LK (p < 0.05), and also no differences from CK and LK (p > 0.05). Among the seven types of flavonoid metabolites of Goji, the content of flavone (Fig. 7B) and anthocyanins (Fig. 7G) showed a decreasing and then increasing tendency with the increase of K supply. Flavone content was affected by K supply (p < 0.05), and it reached the top in HK treatment. The content of anthocyanins was the highest in the LK treatment, which was significantly greater (p < 0.05) than that in the CK and HK treatments. The flavanone content (Fig. 7D) had a positive correlation with K fertilizer supply, and its content in HK level was obviously different from that in LK and CK levels (p < 0.05). The isoflavone content (Fig. 7F) featured an increasing and then decreasing trend with increasing K supply. It was the highest in CK and was significantly different from that in LK and HK treatments (p < 0.05). Just a slight difference appeared in the content of flavone C-glycosides (Fig. 7C), flavonol (Fig. 7E), and flavonolignan (Fig. 7H) in different K levels.Fig. 7 Metabolite ion intensity of flavonoids in Goji fruits. (A) Total flavone content of goji fruits; (B, C, D, E, F, G, H) metabolite ion intensity of flavone, flavone C-glycosides, flavaone, flavonol, isoflavone, anthocyanins and flavonolignan content in Goji fruit; small letters indicate a significant difference among LK, HK, and CK (p < 0.05), as analyzed by Duncan’s multiple tests.

Metabolic profiling of flavonoid in Goji fruits at different K fertilizer levels

Heat map cluster analysis of flavonoid metabolites in Goji fruits was performed (Fig. 8). The results demonstrated that 132 flavonoid metabolites grouped according to the trend of the contents with K decreasing. The red group showed high content while the blue group exhibited low content. As the K contents declined, the color changed from blue to red means that the content of the ingredient was gradually increasing. There was a negative correlation between metabolites and K contents. While the color changed from red to blue means a positive relationship. The heatmap also showed the results of the cluster analysis. Metabolites clustered into one group share the same trend.Fig. 8 Heat map concerning flavonoids of Goji fruit. (A) Anthocyanins and proanthocyanidins of Goji fruit; (B, C) flavanone and flavone of Goji fruit; (D) flavone-C-glycosides and flavonolignan of Goji fruit; (E, F) flavonol and isoflavone of Goji fruit.

In anthocyanins (Fig. 8A), the differences are mainly due to cyanidin O-syringic acid, pelargonidin, delphinidin 3-O-glucoside (Mirtillin), rosinidin O-hexoside, malvidin 3,5-diglucoside (Malvin) and delphinidin 3-O-rutinoside (Tulipanin). In proanthocyanidins (Fig. 8A), the differences were not obvious. In flavanone (Fig. 8B), naringenin, naringenin chalcone, and butein were upregulated with K increasing, while eriodictyol was down-regulated. In flavone (Fig. 8C), the differences are mainly due to tricin O-eudesmic acid, Apigenin 7-O-neohesperidoside (Rhoifolin), Acacetin, and Butin. 2 metabolites in flavone-C-glycosides had significant change (Fig. 8D). One was C-hexosyl-chrysoeriol O-sinapoylhexoside which had obverisly upregulated with rising fertilization. The other one was hesperetin C-hexosyl-O-hexosyl-O-hexoside which had the opposite trend. The result of flavonolignan metabolite showed that the contents of tricin 4′-O-(syringyl alcohol) ether 7-O-hexoside had a positive correlation with K level (Fig. 8D). In flavonol, syringetin 3-O-hexoside had a positive correlation with the level of K, while isorhamnetin 3-O-neohesperidoside and Kaempferol 3-O-rhamnoside (Kaempferin) were significantly downregulated (Fig. 8E). The heat map of isoflavone indicated that the content of 6-Hydroxydaidzein was increasing, yet glycitin was decreasing with rising levels of K fertilization (Fig. 8F).

Discussion

The research about the effects of K on the agronomic traits and fruit quality of Goji was fewer. However, it is necessary to know the relationship between k fertilization and yield qualities, which is important to companies. In this study, the differences in agronomic traits and nutrients of Goji under three K levels were revealed.

It was found that foliar spraying of KH2PO4 on Lycium ruthenicum Murray significantly increased the yield of a single plant and the weight of 100 fresh fruits. However, it had no significant effects on the longitudinal diameters, transverse diameters, and the fruit type index of black fruits. With the increasing KH2PO4 spraying concentration, the size of black fruit tended to increase and then decrease31. In present study, K levels had a significant effect on the ratio of the longitudinal diameter to the transverse diameter of Goji fruits, and the influence on plant height, plant stem diameter, new branch increment, yield of fresh fruits per plant, dry fruit quantity within 50 g, ratio of different grade fruits, was slight (Figs. 1 and 2). The shape of red fruit was flatter at LK, rounder at CK, and longer at HK. It was entirely different between black and red fruit, mainly because of Lycium ruthenicum Murr. and L. barbarum belong to different varieties and have different genetic backgrounds. Previous research also indicated that both the environment and genotype had a highly significant impact on the differences in fruit shape33.

It showed that the application of K fertilizer had an effect on the improvement of Goji fruit quality. The content of sugar and vitamin C in fruit was higher than that in those fruits without K fertilizer12. However, there were no statistical analyses of the experiment data, and the nutrient content and metabolites were not analyzed. In previous study, K fertilizer had the greater effect on the content of polysaccharides, total sugar, and carotenoids of ‘Ningqi 1′34. Nevertheless, the material was different from this study and the experiment was done in the field, where many uncertainty factors occur. It illustrated that there was a significant correlation between the yield qualities of Goji and the amount of N and K applied. With the increase of N and K, the content of polysaccharides, flavonoids, and β-carotene increasing, while the content of betaine and the yield increased and then decreased35. However, whether the effects on yield or quality of Goji were N or K was uncleared. It has been indicated that the flavonoids content in leaves and fruits of Lycium barbarum L. presented a decreasing and then increasing trend before harvesting. It was dropped to the lowest point when the fruits were ripe28. It was the same trend with the expression level of key genes related to flavonoid synthesis29,30. This experiment showed the trend of flavonoids content before harvesting. The fertilizer influence on nutrients in the total period of harvest, from mid-June to mid-July, was not researched. During the development of Lycium chinense Miller fruits, the trend of flavonoid content was increased with the gradual maturation of the fruit. It was shown to be higher at the late stage of development36. Similarly, the material was different and it did not show the effect of K on nutrients of Goji. Previous studies have shown that genotypes, altitude, soil and climate conditions, ecological factors, fruit ripening levels, and extraction methods have a strong impact on fruit contents33,37,38. In present study, the contents of amino acids, flavonoids, polysaccharides, and betaine of Goji fruit were analyzed under three K treatments at different harvest. It was demonstrated that K fertilization had affected the content of glutamic acid and flavonoids in Goji fruits. The higher the level of K, the higher the content of glutamate but the lower the content of flavonoids (Figs. 3A and 4A). The levels of proline, leucine, valine, glycine, and aspartic acid increased with the improvement of K, while the levels of threonine, alanine, methionine, and tyrosine decreased (Fig. 3B). It was illustrated that the glycine amino acid had a significant impact on the number of runners per main plant and per unit area in strawberry young plants, rather than on runner quality parameters39. More research should be conducted to determine whether amino acids impact the yield and quality of Goji fruits. Furthermore, the effect of K on flavonoid metabolites of Goji fruit was analyzed. The KEGG pathway showed that the flavonoids of Goji were synthesized through the phenylpropanoid pathway (Fig. 5). Using PCA analysis, it was proved that there were large differences among LK, CK and HK (Fig. 6). 132 flavonoid metabolites were identified, of which 36 metabolites up-regulated and 30 metabolites down-regulated. The results presented that the higher the K supply, the lower the total flavanone content but the higher the flavanone content. The content of flavonol and anthocyanins decreased, and then increased with the increasing K application. On the contrary, isoflavone content increased and then decreased with the increase of K application. The content of flavone C-glycosides, flavonol and flavonolignan was not biologically statistically significant (Figs. 7 and 8). Different levels of K fertilization lead to varying nutrient compositions in Goji fruits. This may be linked to the root system's ability to absorb essential elements from the soil and transport them within the plant. The root system's characteristics, such as spreading area, capillary root density, cation exchange capacity, and root secretions, all play a role in this process40–43.

Finally, correlating the results of agronomic traits with nutrient, we found that the fruit picked at the second time had the highest yield, the largest size, and the lower content of total amino acids and betaine, while the fruit picked at the fourth time had the smallest size and the higher content of total amino acids and betaine. It indicated that the total amino acids and betaine content in small ‘Ningqi 7’ fruits were significantly higher than those in large fruits.

Conclusion

The study discovered that the fruit shape of Goji could be significantly changed by K supply. With the increase of K content, the fruit shape changed from flat to round and then to long. K fertilization affected the contents of glutamic acid and flavonoids in Goji fruits. The content of glutamic acid was positively correlated with K supply, while that of flavonoids was negatively related to K supply. The study also presented that even though larger fruits have higher market prices and purchasers, their total amino acids and betaine contents were not as good as smaller ones. The results provided evidence of increasing the quality of Goji fruits, which may influence the hobbies of purchasers.

Materials and methods

Plant materials

The variety is called 'Ningqi 7' and it is three years old as of 2022. The seedlings were provided by the National Wolfberry Engineering Research Center in Ningxia, China. The coordinates of the center are 38°39′ 2.83′′ N, 106°9′15.21′′ E, and it is situated at an altitude of 1107.53 m. The average annual precipitation is approximate 200 mm, the average annual temperature is almost 8 °C, the annual evaporation is about 1595 mm, and the annual sunshine hours are close to 2900 h.

Experimental design and conditions

The experiment was conducted in the phytotron of the Wolfberry Scientific Research Institute base of Ningxia Academy of Agricultural and Forestry Sciences, Ningxia, China. The temperature ranged from 24–27 °C and the humidity ranged from 70 to 80%. The illumination intensity ranged from 110 to 125 μmol m−2 s−1 (14 h light, and 10 h dark). There were fifteen plants assigned to each treatment, which planted in pots (diameter: 25 cm, height: 50 cm) containing 5 kg soil, respectively.

The measurements of soil conditions were as follows. The soil PH was detected using a DDSJ-319L electrode pH meter. In addition, total N (TN), total P (TP), total K (TK), available N (AN), available P (AP), available K (AK), total salt (TS), and soil organic matter (OM) were evaluated by using the standard test methods of the Chinese national standard44. Therefore, the conditions of the soil were: pH, 7.84; EC, 0.068 dS/m; TS, 0.42 g/kg; OM, 5.12 g/kg; TN, 0.44 g/kg; TP, 0.72 g/kg; TK, 7.50 g/kg; AN, 23.00 mg/kg, AP, 12.00 mg/kg; AK, 15.00 mg/kg.

Five trees were randomly assigned to each treatment, which had three replications. As the study of H45, every year 500 g of fertilizer was applied per plant to produce 500 g of dry fruits. The N, P and K fertilizer were supplied as urea (N 46%), ammonium dihydrogen phosphate (N 12%, P 61%), and potassium sulfate (K 52%), respectively. Based on the calculations, the total contents of N, P, and K elements were 200 g/plant. According to the previous research 45–47, the control treatment was N, 85 g/plant; P, 65 g/plant; K, 50 g/plant. The three treatments in this experiment were, low K (LK) 25 g/plant, high K (HK) 75 g/plant, and control K (CK) 50 g/plant, as shown in Table 1. The total fertilizers of Goji were split into 3 times a year, in late March, early June and mid-August. For N, based fertilizer was 60%, then top dressing 20% for twice. For both P and K, based fertilizer was 40%, then top dressing 30% for twice. Each time, mixed fertilizers with water and supplied them to the pot.Table 1 Experimental treatments.

Treatment	Nitrogen (g/plant)	Phosphorus (g/plant)	Potassium (g/plant)	
LK	85	65	25	
HK	85	65	75	
CK	85	65	50	

Measurement of agronomic traits of Goji under different K treatments

Measurements of seven agronomic traits in three groups are shown as follows. In April, the plant height and the plant stem diameter at 5 cm above the ground were measured with the tape measure and vernier caliper respectively. In the middle of May, when the new branches grew to about 5 cm, a branch of each tree was selected and marked. Every 5 or 6 days, its length on the marked new branches was measured with a tape measure for a total of 5 times. From mid-June to mid-July, ripe Goji fruit was picked four times. At each time, the fresh fruits from each tree were picked, packed, and weighed separately. In the meanwhile, 5–6 fresh fruits were packed in 10 ml freezing tubes and stored in liquid nitrogen (− 196 °C) rapidly, then keep the samples in a refrigerator at − 80 °C to detect metabolites. At the same time, 5 fruits of each treatment when harvested were randomly selected, and their length and transverse diameters were measured. After that, the fresh fruits were transported to the drying room immediately at each time (50 °C for 5 h) and then preserved the samples in a dry and ventilated room to be counted, weighed, and graded. In late July, all the dry fruit samples from each time were prepared. The number of fruits within 50 g was counted before being graded. Because it is necessary to know whether K treatments influence fruit size, relating to the market prices of Goji closely. Finally, graded the dry samples using 3 sieve sizes: the first grade was 180 fruit/50 g; the second grade was 220 fruit/50 g; and the third grade was 280 fruit/50 g. The ratio of the different grades was measured.

Nutrient composition of Goji fruit

For each grade the composition of polysaccharides, amino acids, total flavonoids and betaine contents were measured.

Dry fruit samples were freeze-dried using a vacuum freeze dryer. Then cut the samples and freeze them using liquid nitrogen. Adding PBS after melting and homogenizing samples to pulp. Then centrifuged for 20 min and removed supernatant. To detect polysaccharides, total flavonoids, and betaine contents, the plant polysaccharide test kit, the plant flavonoid test kit, and the plant betaine test kit were applied separately46,48.

To detect amino acid contents, crushed samples were prepared. 0.02 mol/L hydrochloric acid was added, then centrifugation for 10 min. The supernatant added 6–8% sulfosalicylic acid. Then centrifuged for 15 min and obtained supernatant. The liquid samples were passed through the film on the machine, which obtained amino acid contents46.

Quantification of flavonoid metabolites

Fresh fruit samples were freeze-dried, then crushed using a mixer mill. Added 70% aqueous methanol and centrifugation for 10 min. The extracts were absorbed and filtrated, then analyzed using an LC–ESI–MS/MS system. The HPLC conditions, including HPLC column, solvent system, gradient program, flow rate, and injection volume, were regulated to the appropriate state46,47. Mass spectrometry was used to elute metabolites and perform multiple reaction monitoring (MRM) experiments49. The qualitative analyses of metabolites was based on the self-built database MWDB (Metware Biotechnology Co., Ltd. Wuhan, China) and the public database of metabolite information. The quantitative analysis of metabolites was performed using MRM analysis of mass spectrometry50–52.

Statistical analyses

The data for agronomic traits and nutrient content were analysed using Microsoft Office Excel 2016, SPSS 23.0 (IBM Corporation, Armonk, NY, USA) and Graphpad Prism 8.0 (Graphpad software, Inc., 7825 Fay Avenue, Suite 230, La Jolla, CA 92,037 USA). The results for the flavonoid metabolites were analyzed by using R (http://www.r-project.org/), which performs orthogonal signal correction and partial least squares-discriminant analysis (OPLS-DA)53.

Acknowledgements

The authors gratefully acknowledge the financial support (32160062, 2021BEF0200101, 2023AAC03399 and 2018NYYZ0106) of the Ningxia Hui Autonomous Region.

Author contributions

R.W., Z.S. and Y.L. wrote the main manuscript text; T.H., Y.C., W.A. and X.Z. prepared Figs. 1–4; J.Z. and K.Q. prepared Figs. 5–8; X.W. and L.Y. investigative data. All authors reviewed the manuscript.

Data availability

The datasets generated and/or analysed during the current study are not publicly available due to all the data are collected by the author, so it is not convenient to disclose but are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Ethical approval

Experimental research and field studies on cultivated plants, including the collection of plant material, comply with relevant institutional, national, and international guidelines and legislation. All methods were carried out in accordance with relevant guidelines in the method section. Authors complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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