Soybean responses to nutrients deciency, and the possibility of detecting this deciency using chlorophyll uorescence technique

Aims Managing plant nutrition is a key factor to getting optimum yield quantity and quality. Soybean is an important plant as an oil and protein producer crop as well as a biological nitrogen xing plant. The aims of current work were studying soybean's responses to some macro and micronutrients deciency stress as well as the possibility of diagnosing this deciency using chlorophyll uorescence technique. Methods The two-year eld experiment during 2019 and 2020 growth seasons were conducted based on a randomized complete block design with three replications. Treatments were use and non-use of N, P, Fe, and Mo, accompanied with and without humic acid. N and P were applied in the soil, but Fe, Mo, and humic acid were foliar applied at the nal vegetative growth stage. Results Results showed that the effect of fertilizer treatments was signicant on all traits. N-P-Fe-Mo improved grain yield and photosynthesis rate, but their application accompanied with humic acid induced a synergistic effect, and the maximum grain yield and photosynthesis were recorded in the N-P-Fe-Mo + HA. Fertilizer application decreased F 0 and F m and increased F v /F m . Besides, there was a signicant negative correlation between leaf's N, P, Fe, and Mo content with F m ; meanwhile, the negative correlation between leaf's nitrogen and F m was stronger than the other applied nutrients (r=-0.767). Conclusions Research ndings show that, it is possible to use the chlorophyll uorescence technique as a valid non-destructive physiological indicator and a quick way to monitor the nutritional status of soybean plant about N-P-Fe-Mo to timely fertilizing. Although soybean is a nitrogen xing plant, but it needs complementary N fertilizer to achieve maximum PSII eciency, minimum chlorophyll uorescence, and optimal yield.


Introduction
Soybean (Glycine max L.) is one of the staple crops throughout the world that plays an important role in public health due to its oil and protein composition (Chen et al. 2005). In developing countries, where the costs for import oils, oilseeds and protein products are high, improving the e ciency of oilseed cultivars is of great importance (Akbari and Soltani 2018). The area under soybean cultivation in Iran in 2019 was 67,000 ha with an average yield of 2388 kg/ha. Meanwhile, the total world cultivated area of this crop was 120501628 ha with an average production of 2769 kg/ha (FAO 2019).
Chemical fertilizers are typically used through soil or foliar application. Leaching, runoff and evaporation, usually decrease nutrients availability for the plants resulted in its de ciency. Therefore, using new technologies, the plant's need for elements should be met promptly and without wasting and polluting the environment (Ramesh et al. 2010).
Micronutrients have many effects on plant performance. These nutrients are involved in many plant's biochemical reactions while participating in the structure of some organs. De ciency of these elements can sometimes inhibit the uptake of other nutrients and growth; therefore, it should be paid more attention to their application. Iron and molybdenum increase the soybean quantitative and qualitative yield by improving growth indices (Rahman et al. 2010). Iron is an essential nutrient for all organisms; its de ciency exists in many crops. The soil iron content is usually high, but a large part of it is xed in the soil as Fe 3+ , especially at high pH, which is not available for the plants (Mimmo et al. 2014).
Iron compounds are the best solution to eliminate iron chlorosis in all soils, especially in alkaline ones, which can cure the most severe iron nutritional problems in plants. The role of Fe in nitrogen xation and the activity of some enzymes such as catalase, peroxidase and cytochrome oxidase has been well established (Askary et al. 2020). Joorabi et al. (2020) reported that nano zinc chelate (ZnO) signi cantly increased proline content, catalase and peroxidase activities and oil yield of soybean under drought. Fe chelate foliar application affects the soybean's leaf area, the number of pods per plant, seeds per pod and 100-seed weight under drought stress (Vaghar et al. 2020). It has been reported that molybdenum foliar application at the 6-leaf stage could increase the mung bean's agronomic and quality indices (Shohlibor Rodgazi et al. 2021).
Photosynthesis, as the main plant metabolism process, strongly is in uenced by the environmental conditions. It consists of four stages: light perception, electron transfer, energy xation, photoassimilate biosynthesis and transfer (Blankenship 2014). It is a major determinant of plant growth and yield; photosynthesis permanence under environmental stresses is important for yield stability. The nutrients increased photosynthesis rate because of improvement of plant growth conditions (Guo et al. 2019). Nutrients de ciency strongly affect the structure and function of the photosynthetic apparatus (Schlau-Cohen and Berry 2015).
Chlorophyll uorescence uses as a method to study disorders in photosynthetic systems. It is a non-destructive tool for estimating the photochemical e ciency and photosynthetic status of plants and has been widely used to evaluate the plant's response to environmental stresses. It is a reliable method for studying photosynthetic processes under environmental stresses (Jin et al. 2015). Using this chlorophyll uorescence technique is rapid and nondestructive that represents thylakoid membrane integrity and the relative e ciency of electron transfer from PSII to PSI (Kalaji et al. 2017) .
Analysis of chlorophyll uorescence of soybean leaves under the ooding period and different nutritional diets showed that maximal quantum yield of PSII photochemistry (F v /F m ) decreased during ooding stress and nitrogen de ciency (Khadempir et al. 2015). It has been reported that the application of 0.9 g/L iron nano-oxide at the boot stage reduced F v /F m of PSII in wheat under dry-land conditions (Narimani et al. 2018).
Optimal plant nutrition promote achievement of maximum quantitative and qualitative yield. The objectives of this experiment were study of soybean's photosynthesis, chlorophyll uorescence and grain yield to de ciency of some important nutrients, as well as studying the relationship between leaf's nutrients contents with chlorophyll uorescence parameters to nding possible detecting nutritional stress using chlorophyll uorescence technique.

Experimental location and design plant material
The experiment was carried out for two consecutive years (2019 and 2020) in the research farm of Lorestan University, with a position of 33º 26' 15'' N, 48º 15' 39'' E, and an altitude of 1117 m above sea level. Before the start of the experiment, 15 random soil samples were taken (0-30 cm depth) by auger, and the samples were combined. Then the combined sample was analyzed ( Table 1). Some of the important climatic parameters were recorded during the experiment period and presented in Table 2. Each year, the experiment was performed as a randomized complete block design with 12 treatments (Table 3) and 3 replications. The amount of fertilizers was determined based on the results of the soil test (Table 1). Nitrogen 150 kg/ha (75 kg at tilling and 75 kg/ha as top dressing at V2) of urea fertilizer (NPK, 46-0-0) and phosphorus 80 kg/ha of triple superphosphate (46% P2O5) were applied before planting and incorporating into the soil by tilling.
Each experimental plot area was 15 m 2 including ve furrows. The distance between the main plots was 3 m and the distance between the blocks was 5 m. The soybean's seeds (Glycine max L. CV. Kosar) 58 kg/ha were inoculated by Bradyrhizobbium japonicum and planed at 4-5 cm soil depth on 28 May each year. The plant density was adjusted at 35 plants per m 2 (P * P = 5.5 cm and R* R = 60 cm) after seedling establishment. A tape irrigation system was used, and the plant water requirement was calculated based on the lack of soil moisture relative to eld capacity. The weeds control was done manually if needed.
Kosar soybean cultivar was released in 2015 as an early-maturing and indeterminate cultivar with averages of 1000seed weight 135 g, oil content 22%, protein content 37%, growth period 110 days, grain yield 3300 kg/ha, and resistant to Phytophtora, loading, and grain shedding.
Spraying was done at 8 am for three consecutive days, i.e. molybdenum on the rst day, iron chelate on the second day and nally, humic acid on the third day. Plastic guards were used between the plots to prevent diffusion to other plots. UK; light intensity 3000 µmol photon m − 2 S − 1 ) during 10 am to 12 pm. In a way, at each plot, two plants and from each plant, three developed leaves (in the upper, middle and lower part of the plant) were randomly selected, then these parameters were measured. Air temperature was 32.4-36.8 ºC. The leaves were placed in the dark for 20 minutes using a leaf clips (Kalaji et al. 2014). The minimum uorescence (F 0 ) with all PSII open reaction centers and the maximum uorescence (F m ) with all PSII closed reaction centers were determined on leaves adapted in the dark (Genty et al. 1989).
The parameter F v /F m was calculated according to the following equations: is the maximum quantum e ciency of PSII in the dark-adapted state; F m is the maximum uorescence (dark); F 0 is the minimum uorescence (dark); F v is the variable uorescence (dark) (F m -F 0 ).

Chlorophyll contents
Leaf chlorophyll content was measured at the early R1 stage and from the youngest mature leaves according to the Arnon method (Arnon 1949). Half a gram of fresh leaf was crushed and ground in a Chinese mortar using liquid nitrogen. Then 20 ml of 80% acetone was added to it and centrifuged at 10000 rpm for 10 minutes. The supernatant was used for spectrophotometry, and the absorbance was read separately at 663 and 645 nm for Chl a and Chl b, respectively. Chlorophyll content was calculated by the below equations.
Where V: nal volume of chlorophyll extracted in 80% acetone; W: fresh weight. Photosynthesis rate Photosynthesis rate was recorded at owering stage (R1) using IRGA photosynthesis device (model LCA4 made by ADC BioSciversity Ltd. UK) at 10 am to12 pm. Leaf analysis for N, P, Fe, and Mo One day after chlorophyll uorescence measurement, leaves that had been used for uorescence measurement and some others leaves that were similar to them in position and development stage were sampled. Leaves were washed with distilled water to remove probable dusts, then with hydrochloric acid (0.1 M), and nally with distilled water again, and oven-dried at 65 ºC for 48 h and then, powdered by a mixer.
Leaf dry weigh (0.2 g) was used for N measuring by Kjeldahl method according to Bremner (1996) using an auto Kjeldahl distiller instrument (K9840 model, Hanon, China).
Half of a gram of dry weight was used for dry combustion (550 ºC, 4h). A 10 mL of 2 M HCl was added to ash and ltered by lter paper. Then its volume increased to 100 mL by distilled water. Fe and Mo concentration in the extraction was determined by an atomic absorption spectrometer (Agilent 240FS AAS, USA). Phosphorus was measured according Chapman and Pratt (1961) using a UV-VIS spectrophotometer (MPADA CO. China) at 640 nm.
Grain yield At harvest maturity (22-25 September each year), a square meter including four middle rows of each plot was harvested. At rst, their grains (with 13% moisture content) were weighed and considered as grain yield. Then, the plant samples were oven (24 hours at 75°C) dried to obtain biological yield.
Data analysis MSTAT-C software was used for data analysis. Bartlett's test was used for testing homogeneity of variances, and means comparison was done by Duncan's multiple range test.

Chlorophyll uorescence
The results showed that fertilizer treatments had a signi cant effect on the chlorophyll uorescence parameters (Table 4, p ≤ 0.01). The highest and lowest F 0 and F m were observed in control (nutritional de ciency) and N-P-Fe-Mo + HA treatment, respectively (Table 5). The plant has shown higher F 0 and F m under nutrients de ciency. However, the applying of all considered nutrients decreased these parameters, which indicated that the nutrition stress was relieved. There were negative and signi cant correlations between leaf's N, P, Fe, and Mo content with F 0 and F m (Table 10). The highest F V and F V /F m were observed in N-Fe-Mo + HA treatment and the lowest ones in control (Table 5). F V /F m was more sensitive to N than the other nutrients (Table 5). Table 4 Compound ANOVA (mean of squares) of the effects of fertilizer treatments on soybean's chlorophyll uorescence parameters.

Photosynthesis rate
Effect of fertilizer treatments was signi cant (Table 6, p ≤ 0.01) on photosynthesis rate. The maximum photosynthesis rate (9.72 µmol Co2 m − 2 S − 1 ) was observed in N-P-Fe-Mo + HA; however, there was no signi cant difference between this treatment and N-P-Mo (Fe de ciency) + HA, and N-P-Fe (Mo de ciency) + HA. The minimum photosynthesis rate (2.62 µmol Co2 m − 2 S − 1 ) was recorded in control. Photosynthesis rate was most sensitive to nitrogen de ciency (Table 7). Chlorophylls content Chl a and Chl b contents were affected by fertilizer treatments signi cantly (Table 6, p ≤ 0.01). The highest Chl a (11.13 mg/gm FW) and Chl b (4.44 mg/g FW) contents were recorded in N-P-Fe-Mo + HA treatment and the lowest ones in control, respectively (Table 7).

Leaf nutrients (N, P, Fe, and Mo) content
Fertilizer treatments affected leaf's N, P, Fe, and Mo meaningfully (Table 6, p ≤ 0.01). Full nutrition (N-P-Fe-Mo + HA) induced maximum leaf's N, P, Fe, and Mo contents. The lowest contents of these nutrients were in control ( Table 7). Application of each nutrient resulted in increased content of the same one in the leaf. Besides, the application of some of these elements had an indirect effect on the others. For example, Fe de ciency resulted in decreased N and Mo, even under application of N and Mo (Table 7). There was a strong negative correlation (r= -0.806**, Table 10) between P content and F m .

Grain yield
Fertilizer application improved the grain yield, so the highest (1772 kg/ha) and lowest (933 kg/ha) grain yield were observed in N-P-Fe-Mo + HA treatment and control, respectively (Table 9). Although complete fertilizer treatment (N-P-Fe-Mo) increased the grain yield signi cantly, but, the one by one removal of iron, phosphorus and molybdenum from N-P-Fe-Mo treatment composition did not cause a signi cant change in yield, however, grain yield was more sensitive to N, Fe, P and nally to Mo, respectively. Interestingly, the use of humic acid alone did not have a signi cant effect on grain yield, but when used with other elements, it improved the effect of those treatments on grain yield (Table 9). Humic acid was able to compensate for the de ciency of phosphorus, iron and molybdenum in treatments that did not have these fertilizers, but could not be a substitute for nitrogen (Table 9).

Biological yield
The results showed that fertilizer treatments had a signi cant effect on biological yield (Table 8, p ≤ 0.01). The highest (5157 kg/ha) and the lowest (3660 kg/ha) biological yield was observed in N-P-Fe-Mo and control, respectively (Table 9). Among the applied nutrients, Mo de ciency had a minimum effect on biological yield. The HA had no signi cant effect on biological yield; meanwhile it was more effective on the grain yield when Mo was removed from the treatment combination (Table 9).  Table 9 Effect of fertilizer treatments on soybean's grain and biological yields.

Discussion
F0 indicates the uorescence level that the Quinone A (QA) acceptor is at its highest oxidation state (PSII center is open). The lower the F0, the better the photosynthetic activity. However, a higher F0 value indicates damage to the PSII electron transfer chain due to a decrease in QA capacity and lack of its complete oxidation. Therefore, under nutritional stress conditions (C), PSII is not work properly. Reaching chlorophyll uorescence to F m is caused by the photons absorbttion and the reduction of all electron carriers, and the closure (saturation) of all reaction centers. When all PSII reaction centers are closed, a gradual increase in uorescence and a decrease in the rate of photochemical reactions occur (Maxwell and Johnson 2000). The F v indicates the reduction situation of the electron acceptor (QA). Chlorophyll uorescence is high when the electron acceptors are in full reduction state, so F v is high, but when the electron acceptors are oxidized, the uorescence value is minimal and the F v value decreases (Zlatev and Yordanov 2004).
The F v /F m is an effective tool to detect damages in the photosynthetic apparatus before these damages been appear in plant morphology; furthermore, it is a good indicator of detecting photoinhibition (Kalaji et al. 2014). It has been reported that the photochemical e ciency of PSII and the activity of PSII reaction centers decreased, and photoinhibition of PSII occurred due to nitrogen starvation (Zhao et al. 2017).
The decrease in this index can be due to photooxidation and damage to the PSII reaction centers. The researchers reported that the F v /F m ratio was 0.8 in the non-stress conditions and values less than 0.8 indicated the existence of biotic and abiotic stresses in plants (Kalaji et al. 2018). Eisvand et al. (2018) reported that using phosphate biofertilizer in the soil + foliar application of Zn improved F v /F m under late-season heat stress and normal conditions resulted in increased wheat grain yield.
Any stress can inhibit electron transfer in the PSII, thus reducing photosynthetic e ciency and increasing chlorophyll uorescence. Nutrient de ciencies impair the function of the photosynthetic apparatus. This de ciency causes some damages to the photosynthetic apparatus by reducing the PSII quantum e ciency. Lack of nutrients causes stress and increases F 0 and F m parameters, which results in reduced PSII quantum e ciency (Kalaji et al. 2018). Nitrogen de ciency reduces the PSII quantum yield and maximal e ciency. Nutrient limitations such as P, K, Ca, Mg, S and Fe also impair the function of the photosynthetic apparatus and reduce the PSII e ciency (Kalaji et al. 2017). Also, reduced PSII e ciency due to insu cient N may be related to the decreases in chlorophyll content (Table 7). In addition, the chlorophyll molecule contains N, making this element an important factor in the development of the photosynthetic apparatus and prevent leaf senescence (Bassi et al. 2018).
The soybean's F v /F m has been studied using nitrogen (urea fertilizer) and bacterial inoculation (Bradyrhizobium japonicum) treatments under waterlogging conditions. Results showed that in normal conditions, the highest F v /F m has belonged to the bacterial inoculation treatment; however, nitrogen fertilizer application caused the highest PSII under waterlogging stress (Khadempir et al. 2015). Reduction of F v /F m under N de ciency may be related to the positive role of N in photosynthesis, which is linked to nitrogen (N) partitioning in photosynthetic enzymes, pigment content, and total number and composition of chloroplasts (Bassi et al. 2018;Marschner 2011;Taiz and Zeiger 2010).
Decreased photosynthesis is mainly due to stomatal (reduced stomatal conductance) and non-stomatal (structure and function of photosystems and Kelvin cycle) factors. Restriction of photosynthesis will increase chlorophyll uorescence (Taiz and Zeiger 2010).
The nutrients amount in the rhizosphere can have special effects on the rate of photosynthesis. It has been reported that the humic acid in both foliar and soil applications could improve the photosynthetic indices of sun ower (Heidari et al. 2020). Also, Zaremanesh et al. (2019) showed that the use of humic acid as a soil application in a pot experiment increased the photosynthesis rate in Satureja Khuzestanica under control and salinity stress. This nding is consistent with our results; however, we were nding a synergistic effect when HA and chemical fertilizer were applied together (Table 7).
Higher concentrations of chlorophylls were observed under nitrogen fertilizer treatment. This result is consistent with the ndings of the other study (Cendrero-Mateo et al. 2015). However, other applied elements did not have a signi cant effect on chlorophylls content, and the use of humic acid alone was not signi cantly different from the control (Table 7). Nevertheless, iron and manganese foliar application on mung bean under dehydration resulted in increased chlorophyll and carotenoids (Izadi and Modares Sanavey 2018) .
Stresses can reduce chlorophyll index (SPAD number) through its degradation and nally decrease net photosynthesis (Liu et al. 2018). Our results showed an improvement in chlorophyll content under nutrients utilization (Table 7). Although the use of nitrogen had a greater effect on chlorophyll concentration than other elements, using all of them plus humic acid (N-P-Fe-Mo + HA) produced the highest amounts of chlorophylls (a and b). In addition to nitrogen, which directly and indirectly affects chlorophyll biosynthesis, other elements are also indirectly involved in its synthesis; P through ATP and phospholipid synthesis, also increasing magnesium uptake; and Mo via improvement the plant's N content by involving in the nitrogen xation process (Marschner 2011). One of the highlighted roles of Fe in the biosynthesis of chlorophyll relates to Chl precursors biosynthesis, where special emphasis is placed on the involvement of iron in the formation of δ-aminolevulinic acid (ALA), the initial committed step in chlorophyll formation (Pushnik et al. 1984).
Application of each nutrient resulted in increased content of the same one in the leaf. Besides, the application of some of these elements had an indirect effect on the others. For example, Fe de ciency resulted in decreased N and Mo, even under application of N and Mo (Table 7). This may be related to Fe's role as an enzyme metal cofactor of the nitrogen's reductive assimilatory pathway such as nitrate reductase and increased its activity (Borlotti et al. 2012;Marschner 2011).
There was a strong negative correlation (r= -0.806**, Table 10) between P content and F m . Therefore, soybean phosphorus status can be monitored by uorescence chlorophyll. Because P is involved in the transformation of energy, regulation of several enzymatic activities (Schulze et al. 2006), biosynthesis of nucleic acids, proteins, lipids, sugars and adenylates (Zhang et al. 2014), its de ciency will affect photosynthesis reaction and is re ected in Chl uorescence.
Iron chelate foliar application on soy increased grain yield (Joorabi et al. 2020). Caliskan et al. (2008) reported that nitrogen and Fe fertilizers had a positive effect on growth parameters and soybean's grain yield.
Mo chelate foliar application at the 6-leaf stage improved the yield and its components in mung bean. Also, it was observe that the nitrogen application reduced the loss of owers and pods and increased the number of seeds per plant resulted in increased yield (Shohlibor Rodgazi et al. 2021), which is consistent with the results of other researchers.
There is a cross-talk between nutrients uptake and metabolism in plants. Therefore, we observe the highest grain yield in full treatment. This is due to the positive function of these nutrients that improve the effectiveness of each other. Nitrogen increases yield by developing leaf area, increasing chlorophyll content, biosynthesis of important enzymes involved in photosynthesis, and preventing leaf ageing (Marschner 2011). Phosphorous improved the grain yield via improved biosynthesis of nucleic acids, proteins, lipids, sugars and adenylates (Zhang et al. 2014). Iron is a vital constituent of electron chains and a cofactor of many enzymes. It is involved in some metabolism processes such as photosynthesis, respiration, DNA synthesis, and also nitrogen xation (Schmidt et al. 2020). Mo is essential for plants as required by several enzymes that catalyze key reactions in nitrogen assimilation, purine degradation, phytohormone synthesis, and sulphite detoxi cation. Moreover, a tight connection between molybdenum and iron metabolisms is presumed (Bittner 2014) .
Iron chelate increases plant biological function due to its ease of uptake by plants (foliar application) and its important roles in plant physiology (Taiz and Zeiger 2010). Iron de ciency will lead to the yellowing of young leaves and a signi cant reduction in photosynthetic activity, resulting in reduced biomass production (Marschner 2011).
The biological yield was increased due to the application of these elements. They develop a root system and energy transfer (P roles), promote protein and enzyme synthesis, photosynthesis and plant growth (N roles), play a vital role in enzymatic reactions and nitrogen metabolism (Fe roles), and nally improve nitrogen xation (Mo role) (Marschner 2011).
De ciencies of nitrogen, phosphorus, iron and molybdenum signi cantly limit the vegetative growth and fertility of legumes. Azizi et al. (2017) reported that the biological yield of the Cicer arietinum L. was improved by applying 4 mg/kg molybdenum without using calcium nano-oxide.
In a water limitation regime, spraying 300 mg/l humic acid in periods improved physiological parameters and increased biological yield in wheat (Tour and Shokuhfar 2019). However, we did not observe a signi cant effect of molybdenum on biological yield, which could be due to low dose and frequency of spraying or time of application (V7, end of vegetative growth).

Conclusion
Chlorophyll uorescence is an estimate of electron transport in photosynthesis, and re ects photosynthesis e ciency. Its monitoring can be a good indicator of changes in the photosynthetic apparatus. Plants show different photosynthetic characteristics under different environmental conditions such as nutrients availability and biotic and abiotic stresses. These stresses can be traced using chlorophyll uorescence technique even before the onset of their general morphophysiological symptoms. In the current research, fertilizer treatment had a signi cant effect on all studied traits. Measurement of chlorophyll uorescence parameters before owering at the beginning of reproductive phase (R1) showed that F 0 and F m parameters increase when fertilizers are not applied, and PSII photochemical performance is improved by fertilizers application, especially nitrogen.
There was a signi cant relationship between the content of these elements (N-P-Fe-Mo) in the leaves with chlorophyll uorescence parameters, which can be a basis for using these parameters as a valid, non-destructive and rapid physiological indicator for detecting nutrient de ciencies in soybeans in precision agriculture resulting in optimum yield. Of course, this work requires further research on the integration and modelling of uorescence parameters along with growth stages, soil properties and other plant appearance characteristics.