Effect of the concentration and ionic form of nitrogen (N) on photosynthesis, growth and fruit production of blueberry ( Vaccinium corymbosum L.)

Efecto de la concentración y forma iónica del nitrógeno (N) en la fotosíntesis, crecimiento y producción de frutos de arándano (Vaccinium corymbosum L.)

Raúl Cárdenas-Navarro

Universidad Michoacana de San Nicolás de Hidalgo, Mexico

Jesús Alonso Luna-Béjar

Universidad Michoacana de San Nicolás de Hidalgo, Mexico

Vilma del Carmen Castellanos-Morales

Universidad Michoacana de San Nicolás de Hidalgo, Mexico

Nayda Luz Bravo-Hernandez

Universidad Michoacana de San Nicolás de Hidalgo, Mexico

Luis López-Pérez luis.lopez.perez@umich.mx

Universidad Michoacana de San Nicolás de Hidalgo, Mexico

Nitrogen (N) is considered the most important mineral nutrient for plants, it is found in a higher proportion than other essential elements (1 % to 3 % dry matter), depending on the plant species, phenological stage, and the organ. It is part of fundamental molecules for growth and development such as nucleic acids, amino acids, proteins, chlorophylls and alkaloids (Marschner, 2011). Plants have developed physiological mechanisms to uptake mineral N from the soil, mainly in the forms of nitrate (NO₃^-) or ammonium (NH₄⁺) (Errebhi and Wilcox, 1990; Cárdenas-Navarro et al., 2006; Lobit et al., 2007). The N absorbed as NO₃^- is reduced to NH₄⁺ by the enzymes nitrate reductase (NR) and nitrite reductase (NiR). When this process takes place in the leaves, as in most plants, it depends on reductant compounds generated by photosynthesis, and when it takes place in the roots, the reductants compounds are provided by respiration (Scheurwater et al., 2002; Li et al., 2013). The NH₄⁺ absorbed, or previously produced by the reduction of NO₃^- is assimilated directly into amino acids by the enzyme glutamine synthetase (GS) and requires C compounds to get C skeletons and energy, which can come from previously stored carbohydrates or directly from photosynthesis (Li et al., 2013; Doyle et al., 2021). On the one hand, N deficiency directly and negatively affects photosynthesis reducing stomatal conductance, content of light harvesting proteins and content and/or activity of photosynthetic enzymes (Mu and Chen, 2021) and indirectly, due to photoassimilates accumulation, through a feedback downregulation mechanism (Araya et al., 2010). On the other hand, N assimilation consumes carbohydrates originally produced by photosynthesis and competes with other physiological processes associated with plant growth and development for these compounds (Li et al., 2013).

Plants preferences for NH₄⁺ or NO₃^- depend on the species, phenological stage, habitat of species origin and conditions of roots environment such as pH, aeration, temperature (Li et al., 2013). V. corymbosum L. is native from the temperate forests of North America and grows in the undergrowth, where low temperatures limit the mineralization of the plenty organic matter; in soils with low salinity, acidic pH, abundant organic forms of N, as amino acids and proteins and scarce mineral N, which is mainly found as NH₄⁺ (Korcak, 1989; Rosen et al., 1990; Metcalfe et al., 2011). Therefore, this plant species has evolutionarily adapted to grow in soils with low N availability (Banados et al., 2012; Vargas and Bryla, 2015) and to prefer NH₄⁺ instead NO₃^-, which allows it to conserve energy, which is an advantage in a low-temperature environment, since the processes of absorption and assimilation of NH₄⁺:NO₃^- require less energy than those of NO₃^-; in addition, the assimilation of NO₃^- is restricted by the low content of NR in its leaves and roots (Poonnachit and Darnell, 2004; Osorio et al., 2020; Doyle et al., 2021; Leal-Ayala et al., 2021). Indeed, it has been documented that when N is supplied as NH₄⁺, blueberry plants show greater net photosynthesis, leaf area, biomass and content of chlorophyll and mineral nutrients, than when N is provided only as NO₃^- (Osorio et al., 2020; Leal-Ayala et al., 2021; Yuan-Yuan et al., 2021). However, it is unknown if these effects are kept when N availability in the root medium is variable and if there is interaction between the concentration and the ionic form of this element. Therefore, the aim of this work was to study the main effects and the interaction of N concentration and NH₄⁺:NO₃^- proportion in the nutrient solution, on net photosynthesis, plant growth, fruit production and quality of blueberry plants var. Biloxi grown in hydroponics.

The obtained results on PN showed statistically significant effects of N concentration, NH₄⁺:NO₃^- proportion and interaction of both factors (Table 1). The main effects observed is that plants irrigated with 0.6 mM N and those that received this element only as NO₃^- showed lower rate of net CO₂ assimilation (Fig. 1A, 1B). However, when examining factors interaction, it’s clear that the effect of the NH₄⁺:NO₃^- proportion is different in the two N concentrations studied. The PN drop observed with the increase of NO₃^- proportion in the irrigation solution was only showed by plants that received 6.0 mM of N, while in plants irrigated with 0.6 mM of N non statistically significant differences were detected between NH₄⁺:NO₃^- proportion treatments (Fig. 1C).

Table 1
P values from bi-factorial ANOVA analysis of: net photosynthesis (PN), leaf area (LA), shoots fresh weight (SFW), roots fresh weight (RFW), fruit yield (FY), fruit diameter (FD) and degrees brix (ºBrix). The factors were N concentration in irrigation solution ([N]) (0.6 mM and 6.0 mM), and NH₄⁺∶NO₃^- proportion in irrigation solution (NH₄⁺∶NO₃^-) (100 %:0 %, 50 %:50 %, 0 %:100 %); [N] * NH₄⁺∶NO₃^- indicates the interaction of both factors.

Figure 1
Effect of N concentration in irrigation solution (A); NH₄⁺∶NO₃^-proportion in irrigation solution (B) and interaction of both factors (C), grey bars 6.0 mM N concentration and white bars 0.6 mM N concentration on net photosynthesis (PN). Values are the mean ± standard error; different letters in the bars indicate statistically significant differences according with Tukey test at P ≤ 0.05.

The plant biomass production analysis revealed that the variables representing the aerial part growth of plant (the LA and the SFW), showed a similar response that PN. In both variables the effects of the studied factors and their interaction were statistically significant (Table 1), thus, the plants irrigated with 0.6 mM N and those receiving only NO₃^- showed the lowest values (Fig. 2A, 2B, 3A, 3B). Nevertheless, the reduction of LA and SFW observed in plants receiving just NO₃^- in the nutrient solution was only perceived in plants irrigated with 6.0 mM N, whereas non statistically significant differences were detected between NH₄⁺:NO₃^- proportion treatments when plants were irrigated with 0.6 mM N (Fig. 2C, 3C). On the other hand, the RFW did not showed statistically significant effects associated with the studied factors or its interaction (Table 1, Fig. 4A, 4B, 4C).

Figure 2
Effect of N concentration in irrigation solution, (A); NH₄⁺∶NO₃^- proportion in irrigation solution, (B) and interaction of both factors, grey bars 6.0 mM N concentration and white bars 0.6 mM N concentration (C) on leaf area (LA). Values are the mean ± standard error; different letters in the bars indicate statistically significant differences according with Tukey test at P ≤ 0.05.

Figure 3
Effect of N concentration in irrigation solution, (A); NH₄⁺∶NO₃^- proportion in irrigation solution, (B) and interaction of both factors, grey bars 6.0 mM N concentration and white bars 0.6 mM N concentration (C) on shoots fresh weight (SFW). Values are the mean ± standard error; different letters in the bars indicate statistically significant differences according with Tukey test at P ≤ 0.05.

Figure 4
Effect of N concentration in irrigation solution (A); NH₄⁺∶NO₃^-proportion in irrigation solution (B) and interaction of both factors, grey bars 6.0 mM N concentration and white bars 0.6 mM N concentration (C) on roots fresh weight (RFW). Values are the mean ± standard error; different letters in the bars indicate statistically significant differences according with Tukey test at P ≤ 0.05.

When examining fruits production and quality it was observed that FY showed statistically significant effects associated to N concentration, to NH₄⁺:NO₃^- proportion and to interaction of both factors (Table 1). The main effects analysis revealed that FY was higher in plants that received solutions with 6.0 mM N (Fig. 5A) and decreased as the proportion of NO₃^- in the irrigation solution increased (Fig. 5B). However, the factors interaction analysis showed that the effect of NH₄⁺:NO₃^- proportion is different in the two studied concentrations of this element. The plants that received 6.0 mM N had lower FY when this element was provided only in the form of NO₃^-, while in the plants that received 0.6 mM of N, the lower fruits production was observed in treatments with 100 % and also 50 % NO₃^- (Fig. 5C). The FD only showed significant effects associated to NH₄⁺:NO₃^- proportion, but not to N concentration or to interaction of both factors (Table 1). The main effects analysis reveals that plants that received only NO₃^- produced the smallest diameter fruits (Fig. 6B) and as the interaction of the studied factors was not significant, this pattern was replicated in both studied N concentrations (Fig. 6C). The ºBrix of fruits did not shows statistically significant effects associated with N concentration, NH₄⁺:NO₃^- proportion, or the interaction of both factors (Table 1, Fig. 7 A, 7B, 7C).

Figure 5
Effect of N concentration in irrigation solution (A); NH₄⁺∶NO₃^-proportion in irrigation solution (B) and interaction of both factors, grey bars 6.0 mM N concentration and white bars 0.6 mM N concentration (C) on fruit yield (FY). Values are the mean ± standard error; different letters in the bars indicate statistically significant differences according with Tukey test at P ≤ 0.05.

Figure 6
Effect of N concentration in irrigation solution (A); NH₄⁺∶NO₃^-proportion in irrigation solution (B) and interaction of both factors, grey bars 6.0 mM N concentration and white bars 0.6 mM N concentration (C) on fruit diameter (FD). Values are the mean ± standard error; different letters in the bars indicate statistically significant differences according with Tukey test at P ≤ 0.05.

Figure 7
Effect of N concentration in irrigation solution(A); NH₄⁺∶NO₃^-proportion in irrigation solution (B) and interaction of both factors, grey bars 6.0 mM N concentration and white bars 0.6 mM N concentration (C) on degrees brix (ºBrix). Values are the mean ± standard error; different letters in the bars indicate statistically significant differences according with Tukey test at P ≤ 0.05.

Blueberry is considered a plant with low nutritional requirements, particularly N (Banados et al., 2012; Vargas and Bryla, 2015), however, in this work plants that received 0.6 mM N in the irrigation solution showed a lower PN (Fig. 1A). The photosynthesis drop in N-deficient plants is produced by a reduced stomatal conductance, a low availability of energy molecules and light harvesting proteins, as well as reduced content and/or low activity of enzymes involved in carboxylation process (Mu and Chen, 2021). Low photosynthesis in N-deficient blueberry plant is also associated with feedback downregulation mechanism generated by leaf carbohydrates accumulation (Araya et al., 2010; Jorquera-Fontena et al., 2018). In addition to display a decline in PN, plants that received 0.6 mM N also exhibited less development of the plant aerial part, specifically LA and SFW (Figs. 1A, 2A); therefore, it is evident that such a N concentration in the irrigation solution generates a deficiency of this element, which is insufficient to meet the N demand created by plant growth. Moreover, plants that received solutions with 6.0 mM N, together with higher PN, LA and SFW, also showed higher FY (Fig. 5 A, 6A, 7A). In most crops, growth and fruit yield rise with the increase of N rate until to a critical level beyond which more N supply increases do not generate augmentations in these variables and may even decline (Cárdenas-Navarro et al., 2004). In blueberry several works, carried out under different agronomic conditions, have reported N rates, varying between 34 and 93 kg of N ha^-1year^-1, beyond which increases in plant growth and/or fruit yield were not observed (Bryla and Machado, 2011; Banados et al., 2012; Ehret et al., 2014; Vargas and Bryla, 2015; Messiga et al., 2018). In accordance with these reports, the 6.0 mM N treatments of this work were equivalent to 18.4 g plant^-1year^-1, meaning 51 Kg N ha^-1year^-1 if a common planting density of 2,777 plants ha^-1 is considered (Banados et al., 2012; Vargas and Bryla, 2015). Some reports in the literature indicate that in blueberry N supply reduce the fruits size and affect its sugars accumulation (Ehret et al., 2014; Vargas and Bryla, 2015; Zhang et al., 2023); however, in this work N treatments did not generate statistical differences neither in FD (Fig. 6A) nor in ºB (Fig. 7A).

Plants uptake mineral N from the soil mainly as NH₄⁺ or NO₃^-, the preference between these two ionic forms of N depends on plant species, its origin habitat, its phenological stage and the roots environmental conditions such as pH, temperature, aeration and microbial activity (Li et al., 2013). In the literature, most authors claim that blueberry develops better when N is provided only or mainly as NH₄⁺ (Doyle et al., 2021). In this work, such a preference is confirmed according to the responses registered in the evaluated variables. Firstly, plants that received nutrient solutions in the presence of NH₄⁺ showed higher PN (Fig. 1B), compared to those that received only NO₃^-, which agrees with previously published reports on this crop (Osorio et al., 2020; Yuan-Yuan et al., 2021). Secondly, as is well known, photosynthesis is the main physiological process associated to biomass accumulation and crops production; in this work, plants that received nutrient solutions with NH₄⁺, in addition to display higher PN also produced a higher aerial biomass: LA and SFW (Fig. 2B). These results are in accordance with a wide number of previously published reports on blueberry, in which plants biomass increase when supplied with NH₄⁺, alone or predominantly, and decrease when received only NO₃^- (Osorio et al., 2020; Doyle et al., 2021; Yuan-Yuan et al., 2021). This behavior is surely associated with V. corymbosum L. adaptation to its origin habitat, which is the temperate forests undergrowth and soils with acidic pH, low temperatures, low microbial activity, slow process of organic matter mineralization and NH₄⁺ as the predominant inorganic form of N (Korcak, 1989; Metcalfe et al., 2011).

The blueberry plant NH₄⁺ preference reflects its adaptation to such environmental conditions and is expressed in physiological process as absorption, translocation from the root to the aerial part and assimilation in organic N (Doyle et al., 2021). Thirdly, NH₄⁺:NO₃^- proportion effects on PN, LA and SFW were likewise observed on the production and quality of fruits. The FY of plants that received only NH₄⁺ was three-fold than those received only NO₃^- (Fig. 5B), these plants also showed bigger FD (Fig. 6B), although non-statistical differences were observed in ºBrix (Fig. 7 B), which agrees with previous reports (Bolaños-Alcántara et al., 2019). The increases of FY and FD are surely associated with the higher availability of photo-assimilates, as a result of PN increase; the greater development of LA and the higher number of flower buds and consequently of fruits, as outcome of SFW increase of plants that received NH₄⁺ in the irrigation solution.

However, the analysis of studied factors interaction showed that the effect of NH₄⁺:NO₃^- proportion on PN, LA, SFW and FY depends on N availability in roots environment. PN of plants that received irrigation solutions with 6.0 mM N was more than twice higher when this element was supplied as NH₄⁺, than when it was provided as NO₃^-. In plants that received 0.6 mM N, no statistical differences were observed between NH₄⁺:NO₃^- proportion treatments (Fig. 1C). It is well established that N is essential in plants photosynthesis and that when its availability in the root milieu is limited, as is the case of plants that received 0.6 mM N in this work, CO₂ fixation decrease, regardless of the ionic form of N supplied in the irrigation solution. In such a condition, as it has been previously reported by several authors in blueberries, photosynthesis decline, either due to the lack of essential compounds that participate both in the transformation of light energy into energetic molecules and in the carboxylation process, or by the feedback downregulation mechanism exerted by the excessive accumulation of carbohydrates (Araya et al., 2010; Jorquera-Fontena et al., 2018; Petridis et al., 2020; Leal-Ayala et al., 2021; Mu and Chen, 2021). On the other hand, when plant growth is not limited by the N availability in the root medium, as in the case of 6.0 mM N treatments, the development of the photosynthetic process is not obstructed by the lack of N compounds nor downregulated by carbohydrates accumulation in leaves cells. In such conditions, PN showed by plants that received only NH₄⁺ was more than two folds greater than by those supplied only with NO₃^- (Fig. 1C), which could be associated to carbohydrates consumption by NH₄⁺ assimilation processes (Li et al., 2013), stimulating CO₂ fixation, according to the sugar-sensing mechanism previously proposed in blueberry (Jorquera-Fontena et al., 2018; Petridis et al., 2020).

The pattern observed on PN, was similar to plant aerial biomass production both in LA and in SFW. In these variables, plants irrigated with solutions at 6.0 mM N showed six- and five-times greater values, respectively, when N was supplied only as NH₄⁺ than when it was provided just as NO₃^- (Fig. 2C, 3C). Furthermore, when the N concentration in the irrigation solution was 0.6 mM none of these variables exposed statistically significant differences between NH₄⁺:NO₃^- proportion treatments (Fig. 2C, 3C). These results are surely associated with the previously shown behavior of PN, as this physiological process is considered the most important for plant biomass accumulation (Evans and Clarke, 2019; Flood et al., 2011; Yamori, 2020); although probably there are also interactions between the factors and variables studied in this work, with other physiological processes as respiration, water absorption and transpiration.

In blueberry, fruit production depends mainly on the number of plant floral buds (Salvo et al., 2011; 2012; Kumarihami et al., 2021), whose induction is stimulated by factors such as temperature, light intensity and carbohydrates availability (Pescie et al., 2011; Salvo et al., 2012; Kumarihami et al., 2021). Carbohydrates metabolism plays a fundamental role, since it is a source of soluble sugars such as glucose and fructose, which act as energy providers and as signaling molecules that stimulate the metabolic processes involved in breaking bud dormancy (Wang et al., 2021). The first phases of the flower buds induction are sustained mainly by the reserve carbohydrates, and later by fruits photosynthesis, but fundamentally by leaves photosynthesis (Maust et al., 1999a; b). In this context, the production and quality of the fruits is based on a source (leaves) sink (fruit) relationship, whose balance depends fundamentally on the factors that affect the photosynthetic activity of the leaves and the number of fruits (Jorquera-Fontena et al., 2018; Kumarihami et al., 2021). As previously showed, plants that received 6.0 mM N as NH₄⁺, displayed the highest values of PN per unit of leaf surface and in plants that received 0.6 mM N there were no statistically differences between NH₄⁺:NO₃^- proportion treatments (Fig. 1C). The same pattern was observed in LA (Fig. 2C) and in SFW (Fig. 3C), therefore, according to the source-sink approach, it can be assumed that the behavior of LA and SFW was associated with photoassimilates availability.

FY and FD showed similar effects than PN, LA and SFW; plants that received 6.0 mM N as NH₄⁺ were more than three times and 15 % higher, respectively, than those that received it as NO₃^- (Fig. 5C, 6C); therefore, it could be assumed that the effects showed by FY and FD depends likewise on carbohydrates availability (Maust et al., 1999a; b). However, interestingly and in contrast to PN, LA and SFW, plants that received 0.6 mM N did show statistically significant differences between NH₄⁺:NO₃^- proportion treatments in both variables. The FY and FD of plants that received only NH₄⁺ were four times and 9.5 % higher, respectively, than those that received only NO₃^- (Fig. 5C, 6C). These results could be explained by an indirect effect of NH₄⁺ on flower buds induction and fruit development, through a stimulus to the photosynthetic activity and the increase of photoassimilates availability, which may act as energy providers and/or signaling molecules (Li et al., 2013; Jorquera-Fontena et al., 2018; Maust et al., 1999a; 1999b; Petridis et al., 2020; Wang et al., 2021). However, more research is needed to clarify and quantify this process.

The results of this work show that, in blueberry grown under hydroponic conditions, plant growth and fruit production were higher when N was supplied as NH₄⁺, than when it was provided only as NO₃^-. These effects were manifested through higher values of net photosynthesis, leaf area, shoots fresh weight, fruit yield and fruit diameter. However, N concentration in the irrigation solution altered the effect of its ionic form and this pattern was only observed at high N concentration (6.0 mM), whereas at low N concentration (0.6 mM), these effects were only maintained in fruit yield and fruit diameter, but not in net photosynthesis, leaf area and shoots fresh weight. Considering that photosynthesis is a fundamental physiological process for plant growth and development and the intimate relationship between N and C metabolism, it is proposed that the effects of the concentration and the ionic form of N, on growth and fruit production of blueberry plants, were closely related to internal carbohydrates availability.

The present work was carried out as a part of the MSc thesis of Mr. Jesús Alonso Luna Béjar, who was supported by a fellowship from the Consejo Nacional de Humanidades, Ciencias y Tecnología (CONAHCYT) of the Mexican government and was directed by Dr. Raúl Cárdenas-Navarro. Special thanks to M.A in ELT. Mauricio Montes Cortés, from the Languages Department of the Universidad Michoacana de San Nicolás de Hidalgo, for English editing service. This study was funded by the Consejo de la Investigación Científica (CIC) of the Universidad Michoacána de San Nicolás de Hidalgo.