Critical Leaf Magnesium Thresholds for Growth, Chlorophyll, Leaf Area, and Photosynthesis in Rice (Oryza sativa L.) and Cucumber (Cucumis sativus L.)

Abstract

Accurately understanding the critical threshold of leaf magnesium (Mg) concentration is crucial for rapid diagnosis of crop Mg status; however, little information is available on critical Mg concentration for different physiological processes in dicots and monocots. Here, we investigated the sensitivity of biomass, chlorophyll (Chl) at different leaf positions/ages, leaf area (LA), and photosynthesis (P_n) to Mg deficiency between rice (Oryza sativa L.) and cucumber (Cucumis sativus L.). Plants were grown hydroponically under twelve Mg concentration gradients. Results showed reducing the external Mg supply to a certain level resulted in significant decline in biomass, Chl, LA, and P_n in both plants. A leaf Mg threshold of 0.97 mg g⁻¹ DM (dry matter) for total biomass was found in rice, which was not identified in cucumber. Critical Mg thresholds for Chl a, b, and carotenoids (Car) showed a decreasing trend with leaf age, suggesting Chl in upper young leaves are more sensitive to Mg deficiency; however, visible Mg-deficiency symptoms were predominantly in mid-aged leaves with a higher rate of Mg remobilization, especially in cucumber. Leaf critical Mg concentrations for Chl a+b, P_n, and LA were 1.22, 1.05, and 1.00 mg g⁻¹ DM in rice, respectively, which were lower than those of cucumber, 4.23, 4.09, and 3.55 mg g⁻¹ DM, implying that cucumber was more susceptible to low Mg stress; Chl a+b was the most sensitive indicator of Mg deficiency. Overall, Chl a+b of upper young mature leaves can be used as an early diagnostic index of Mg nutrition in crops, especially Mg-insensitive crops.

1. Introduction

Magnesium (Mg), an essential macronutrient for the normal growth and development of higher plants, plays key roles in various biochemical and physiological processes, including chlorophyll (Chl) biosynthesis, photosynthesis, and formation and allocation of carbohydrate [1,2]. A large body of evidence suggests that photosynthesis efficiency and the phloem export of photo-assimilates from source leaves to sink organs are restricted by low Mg supply [3,4,5,6,7,8], consequently resulting in the reduction in photosynthetic leaf area [9] and dry matter of shoot and root [10,11] and a higher leaf mass area [12]. Magnesium deficiency, as one of the most common physiological disorders influencing crop productivity and quality in agriculture [13,14], is becoming an urgent and increasingly serious problem. Thus, to avoid situations of field scale Mg deficiency, it is crucial to have precise knowledge of critical threshold values for Mg concentrations in the plant tissues.

Existing research on Mg deficiency either apply extremely low Mg concentrations or even no Mg, known to certainly induce Mg deficiency, and/or results are not related to the tissue Mg concentration. Furthermore, most studies primarily focus on the physiologic and phenotypic responses of plants to Mg deficiency [5,15,16,17] or optimal Mg ranges for crop growth [18,19]. The concept of critical concentration, which is vital for determining the minimum Mg concentration required for proper plant function, has not been extensively applied in Mg research. Generally, it is considered that the critical Mg tissue concentration for biomass and yield formation is below 1.5 mg g⁻¹ DM [1]. In agricultural practice, the Mg nutrition state needs to be quickly determined in advance to prevent crop yield loss. Leaf diagnosis is a widely utilized technique for assessing plant nutritional status, offering fewer limitations than visual diagnosis. This method determines the nutritional condition of plants by conducting a chemical analysis of their leaf tissue. Usually, the newly mature and fully developed leaf, known as the diagnosis or index leaf, is considered as the most appropriate leaf type, since it is more sensitive to showing the center of the physiological activities [20,21].

Generally, plant intrinsic physiological indices, such as chlorophyll, photosynthesis, etc., may have been affected prior to the onset of visual Mg deficiency symptoms. However, current research mainly focuses on determining the Mg levels or thresholds required for biomass, and some studies suggested this value may not be applicable to physiological parameters. Trankner and Jaghdani [22] proposed that the critical concentrations for photosynthetic capacity in wheat and sunflower leaves were much lower than the reported value of 1.5 mg g⁻¹ DM. Contrasting reports described that critical Mg concentrations for photosynthetic processes were suggested to be higher than those for biomass or harvestable yield [10,23]. Hauer-Jákli and Tränkner [10] re-evaluated data from references and found that critical Mg concentrations for net CO₂ assimilation were higher than those for dry weight in coffee, cotton, rice, and potato. It appears that the sensitivity to Mg availability may vary among different physiological processes. In order to recognize crop deficiency symptoms earlier, we need to find the critical Mg concentrations for growth and the physical processes in crops and screen for the most sensitive indicators of low Mg stress.

Determining nutrient abundance and deficiency via crop visual deficiency symptoms is generally one of the ways to make rapid nutritional diagnoses in the field. As the central atom of chlorophyll, the visual typical symptoms associated with Mg deficiency is interveinal leaf chlorosis [18]. Within a plant, a large proportion (10–35%) of total available Mg is bound to Chl pigments in the light-capturing complex of chloroplasts [1,17,24]. Normally, Mg concentration below 1–2 mg g⁻¹ leaf dry weight is related to the initiation of chlorosis [3,4,19], which is attributed to a decrease in Chl a and b concentrations [4,25,26]. However, there is some controversy about the leaf position where the visual deficiency symptoms initially occur. Previous studies have shown that visual deficiency symptoms typically appear first in older leaves [27,28,29]. Recent hydroponic studies on sugar beet, Arabidopsis, and rice have found that young mature leaves are more susceptible to Mg stress, exhibiting quicker declines in chlorophyll and Mg concentrations compared to the oldest leaves [3,15,30,31]. In field conditions, Zhang et al. [32] observed that the interveinal chlorosis symptom first developed on the leaves above the fruit in wax gourd plants. We inferred that this discrepancy might be related to the different sensitivities of chlorophyll to Mg in leaves at different leaf positions/ages.

Only a few studies have investigated the alterations of Chl pigments at different leaf ages under Mg-deficiency conditions. In citrus seedlings, Mg deficiency was found to decrease Chl a and b and carotenoid (Car) concentration in the middle and lower leaves, while no significant difference was observed in upper leaves [25,26]. Ye et al. [26] reported that Mg deficiency only increased Car/Chl in upper leaves, but the opposite effect was observed in the lower leaves [25]. It is necessary to determine the most sensitive leaf position to low Mg stress to help make more accurate Mg nutritional diagnoses.

Dicots appear more sensitive than monocots in response to Mg deficiency. It is widely accepted that fruits and vegetables (dicots) have a higher demand for Mg than cereals (monocots) in yield [10,33]. A meta-analysis revealed that the recommended application amounts of Mg for fruit, vegetable, and cereal were 94.1, 43.5, and 27.8 kg MgO ha⁻¹, respectively [33]. Meta-analysis has demonstrated that monocots generally have lower critical leaf Mg concentrations for both dry weight (0.07% to 0.16–0.21%) and net CO₂ assimilation (0.02% to 0.41%) compared to dicots (0.10% to 0.70% for dry weight and 0.10% to 0.72% for CO₂ assimilation) [10]. Only a limited number of studies have reported that the sensitivity of photosynthesis in dicots was more sensitive to Mg deficiency than monocots, e.g., sunflower vs wheat [22] and cucumber vs rice [34]. The response between dicots and monocots to low Mg stress exhibits inconsistency within the same physiological process. However, despite these insightful findings, little research is available on the distinct responses between dicots and monocots at a physiological level under varying Mg supply concentrations. Coupled with this, the gradients in previous studies were insufficient, so the critical concentration ranges were vague. In the present study, the sensitivity of biomass, chlorophyll at different leaf positions/ages, leaf area, and photosynthesis were investigated between rice (monocot) and cucumber (dicot) grown hydroponically with twelve Mg supply levels. The main objects of this study were to (1) determine the critical Mg concentration for growth, chlorophyll, leaf area, and photosynthesis of two crops, (2) identify the most sensitive indicators to Mg deficiency, and (3) compare the differences in the response to Mg deficiency between rice and cucumber. Meanwhile, we will analyze the key Mg thresholds for each chlorophyll component at different leaf positions to clarify Mg-sensitive leaf positions as well as chlorophyll types.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The experiment was conducted in a greenhouse with a minimum photosynthetic photon flux density (PPFD) of 1000 mmol photon m⁻² s⁻¹ at leaf level using a 14 h photoperiod at day/night temperatures of 28 °C/20 °C and a constant relative humidity of ~60% on the campus of Nanjing Agricultural University, Nanjing, China. Rice (Oryza sativa L. c.v. ‘Shanyou 63′) and cucumber (Cucumis sativus L. c.v. ‘Jinchun 4′) plants were grown hydroponically in the greenhouse, and the seeds both were bought from the local market.

After sterilization in 10% H₂O₂ for 30 min, rice seeds were germinated in moist gauze. Cucumber seeds were soaked in water for 1 h and transferred to sterile quartz sand for germination. When the seedlings developed an average of 2.5 visible leaves (after a preculture for 10–14 days), the uniform rice (without Mg supply) and cucumber seedlings (with available Mg supply) were transplanted into 6.5 L rectangular containers (30 × 20 × 10 cm, planted with 12 plants) and 1000 mL plastic pots (planted with 1 plant), respectively. At first, seedings were grown in aerated 25% strength Hoagland’s nutrient solution (for composition, see below). Three days later, the seedlings were transferred to a 50% nutrient solution, and after 3 days, seedlings were supplied with full-strength nutrient solution for 3 days. The seedlings were then supplied with full-strength nutrient solution with 12 different Mg concentrations (0, 0.0025, 0.005, 0.01, 0.02, 0.05, 0.10, 0.20, 0.50, 1.00, 1.50, and 2.00 mM). The Mg source was MgSO_4. Nutrient solutions included the following components: macronutrients (mM), 2.86 mM N with equimolar amounts of (NH₄)₂SO₄ and Ca(NO₃)₂; 0.32 P as NaH₂PO₄ and 1.02 K as K₂SO₄; and micronutrients (μM), 35.8 Fe as Fe-EDTA, 9.10 Mn as MnCl₂·4H₂O, 0.52 Mo as (NH₄)₆Mo₇O₂₄·4H₂O, 18.5 B as H₃BO₃, 0.15 Zn as ZnSO₄·7H₂O, 0.16 Cu as CuSO₄·5H₂O, and 0.1 SiO₂ as Na₂SiO₃·9H₂O. CaCl₂ was added to solutions to compensate for the other half of Ca concentration. The nitrification inhibitor, 0.1 mM of dicyandiamide, was added to each nutrient solution to prevent the oxidation of ammonium. The nutrient solutions were changed every 3 days, and the pH was adjusted to 5.50 ± 0.05 for rice and 6.00 ± 0.05 for cucumber daily with 0.1 M HCl and 0.1 M NaOH. All treatments included six replicates in a completely randomized design. The spatial arrangement of the treatments was randomized to avoid edge effects in the greenhouse.

Determination of indicators was performed at 3 weeks after treatments started for rice seedlings and cucumber seedlings.

2.2. Analyses of Gas Exchange

Three weeks after commencement of treatments, leaf gas exchange was measured simultaneously using an open-flow gas exchange system (LI-6400XT; Li-Cor, Lincoln, NE, USA). Measurements were recorded on at least four randomly selected leaves (the first fully expanded leaf) from 09:00 to 14:00 under a light intensity of 1500 μmol photons m⁻² s⁻¹. CO₂ concentration in the leaf chamber (C_a) was set to 400 μmol mol⁻¹ air, leaf temperature was controlled at 27 °C ± 0.2 °C, relative humidity was 50–60%, and the flow rate was 500 μmol s⁻¹. After equilibration to a steady state, gas exchange parameters, including net photosynthesis (P_n), stomatal conductance (g_s), intercellular CO₂ concentration (C_i), and transpiration rate (T_r) were recorded simultaneously.

2.3. Determination of Chlorophyll, Mg Concentrations, and Biomass

After measuring photosynthesis parameters, four tagged plants in each treatment were used to determine physiological characteristics. The leaves of tagged cucumber seedlings were divided into three parts: upper (young), middle (mature), and lower (old) leaves; rice leaves were segmented from top to bottom into four parts: newly fully expanded leaves (L1), the second leaf (L2), the third leaf (L3), and the remaining leaves (Rest). The leaf blade is split along the central vein, with one section dedicated to chlorophyll measurement and the other to magnesium measurement. Chlorophyll concentrations were determined by extracting leaf tissue with 95% (v/v) alcohol [35], followed by dark blanching at 25 °C. The measurements were performed using a spectrophotometer (UV2102; Unico, Shanghai, China) at the absorbance of 665, 649, and 470 nm, and then the chlorophyll and carotenoid concentrations were calculated from those three absorbances. Determination of Mg concentrations was performed with modifications as in Hansen et al. [36]. After being dried to a constant weight at 70 °C, all leaves samples were acid-digested by using 2 mL of 30% H₂O₂ and 5 mL of 65% HNO₃ in a closed-vessel microwave system. After the digestion, Mg concentration in the digestion solution was determined with inductively coupled plasma-mass spectrometry (ICP-MS, NexION 300X, PerkinElmer, Waltham, MA, USA).

The biomass, including roots, stems, and leaves was determined by weighing after oven drying at 105 °C first for 30 min and then at 70 °C to a constant weight for 2–3 days. Leaves were digitally scanned (ES-1200C scanner; Epson, Long Beach, CA, USA), and the area was determined using ImageJ software v1.8.0 (National Institutes of Health, Bethesda, MD, USA). Specific leaf mass (SLM) was calculated by dividing leaf dry matter by leaf area (LA).

2.4. Statistical Analysis

Analysis of variance (ANOVA) was performed using SPSS 18.0 (SPSS, Chicago, IL, USA). The differences between mean values were compared using Duncan’s multiple range test, with p < 0.05 taken to indicate statistical significance. Graphics and regression analyses were performed using Origin Pro 8.5 software and Adobe Illustrator 2020. The responses of biomass, Chls, leaf area, and photosynthetic rate to leaf Mg concentration were described using the linear-with-plateau model.

3. Results

3.1. Phenotypic Changes and Plant Dry Matter Formation

The typical intercostal chlorosis symptoms of Mg deficiency first occurred in the lower old leaves and then extended gradually to the young leaves with the prolongation of Mg-deficiency duration in both rice and cucumber seedlings. Most notably, the basal small older leaves are still green in both plants (Figure 1), while the leaf edges wilt in rice plants. The representative symptoms of Mg deficiency developed in cucumber leaves were significantly stronger than those of rice. The critical Mg supply concentration for cucumber seedlings was 0.2 mM, below which the leaves exhibited symptoms of severe chlorosis, necrosis, and albinism, mainly in the center of middle leaves (Figure 1). Interestingly, the margin had remained green in Mg deficiency symptom leaves for cucumber (Figure 1B,C). Oppositely, Mg deprivation resulted in a visibly dwarfed growth phenotype and marked changes in roots of rice seedlings, especially when Mg supply concentration below 0.02 mM (Figure 1A).

Total biomass of rice seedlings increases rapidly with Mg supply in the range of 0 to 0.02 mM, and then peaks and had a decreasing trend initially when Mg supply increased up to 1 mM. The aboveground, root, and total biomass of rice with the highest values were displayed for the 0.5 mM Mg-treated plant (Figure 2A). The total biomass of Mg-deficient cucumber plants was also decreased, while this trend was not as clearly visible for cucumber compared with rice seedlings (Figure 2B). The root–shoot ratio (R/S) in rice seedlings declined significantly with low Mg supply; however, a trend toward elevated ratio in cucumber was found (Figure 2C).

3.2. Mg Concentration in Leaves at Different Leaf Positions

Reducing the Mg supply greatly reduced leaf Mg concentrations at different positions both in rice and cucumber seedlings (Figure 3). Taking the 2 mM Mg treatment as a control, Mg concentrations of L1, L2, L3, and the rest of the leaves in rice were decreased by 14.0–94.9%, 15.6–96.6%, 19.0–97.2%, and 25.3–87.9%, respectively; Mg concentrations of upper, middle, and lower leaves in cucumber decreased by 9.7–77.3%, 23.1–77.8%, and 0.8–56.5%, respectively (Table S1, Supplementary Materials). When the nutrient solution Mg supply concentration was below a certain value, leaf Mg no longer decreased with decreasing supply concentration values. For example, when the external Mg supply concentration of rice and cucumber was lower than 0.02 and 0.2 mM, respectively, the Mg concentrations at the L2 and L3 leaf position of rice and the middle leaf position of cucumber no longer decreased.

Comparison of different leaf positions, notably, Mg concentration in lower leaves (e.g., rest) of rice, decreased faster initially with decreasing Mg supply, which happened in the middle leaves of cucumber. For example, compared to the 2 mM Mg treatment, leaf Mg concentrations for the 1.5 mM Mg treatment declined by 25.3% and 23.1% in rice rest leaves and cucumber middle leaves, respectively, whereas leaves in all other leaf positions were less than these values (Table S1, Supplementary Materials). Under the sufficient Mg supply condition, the distribution of Mg concentration at different leaf positions showed a trend of L1 < L2 ≤ L3 ≤ the rest leaves for rice and middle < lower ≤ upper leaves for cucumber (Figure 3). Under insufficient Mg supply condition, Mg concentration in lower leaves were higher than that in middle and upper leaves for cucumber; however, no significant difference was found among leaves at different leaf positions for rice, e.g., in the 0.005, 0.01, and 0.02 mM Mg treatments (Figure 3).

3.3. Chlorophyll Concentration at Different Leaf Positions

The concentrations of chlorophyll were highly related with leaf Mg concentration in both rice and cucumber (Figure 4 and Figure S4, Supplementary Materials). The chlorophyll (e.g., Chl a, Chl b, Car, and total Chl) and Mg concentrations at different leaf positions of both plants was fitted well by a linear-plateau model in both plants, except for the lower leaves of cucumber, in which there was extremely notable linear correlation (Figure 4; Table S2, Supplementary Materials). The critical Mg concentrations for chlorophyll among different leaf positions were found. When below the leaf Mg threshold, Chl a, Chl b, Car, and total Chl were reduced significantly with the reduction in leaf Mg concentration. The chlorophyll critical Mg threshold was decreasing with decreasing leaf position in both plants (Figure 4A–F,H), showing that leaves in the upper leaf position may require more Mg to be allocated to chlorophyll to maintain its function compared to the lower leaves. The decline rate of Chl a was higher than that of Chl b and Car, and the Mg threshold corresponding to Car is the lowest at different leaf positions in both seedlings (Figure 4). Both Chl a/b and Car/Chl a+b in rice leaves were significantly negatively correlated leaf Mg. Oppositely, Chl a/b was highly positively correlated with leaf Mg in cucumber (Figure 4I,J).

Chlorophyll a+b in the margins of cucumber leaves was significantly below that in the centers, particularly in high-Mg-supply treatments (Figure S2, Supplementary Materials). The mean Chl a+b in the margins of upper leaves was 21% lower than that in the centers. However, there was no significant difference between leaf margins and centers under low-Mg-supply treatment (below 0.2 mM, Figure S2F), as well as in the middle and lower leaves ignoring treatments (Figure S2E).

3.4. Leaf Area and Specific Leaf Mass

The total LA was significantly reduced by insufficient Mg supply in rice and cucumber seedlings (Figure 5). A significant reduction in LA was observed at a Mg supply below 0.02 and 0.05 mM Mg in rice and cucumber, respectively. The lowest LA value of 48.79 ± 7.34 cm² plant⁻¹ in rice was obtained for the 0 mM Mg-treated group and was 283.24 ± 21.71 cm² obtained with the 0.005 mM Mg treatment in cucumber. Specific leaf mass (SLM) was significantly increased under the conditions of Mg insufficiency in both seedlings, which occurred when the Mg supply was lower than 0.005 mM in rice and 0.02 mM in cucumber, respectively (Figure 5). The mean SLM of rice ranged from 2.41 ± 0.08 to 3.41 ± 0.26 mg cm⁻² and that of cucumber from 0.75 ± 0.15 to 1.60 ± 0.18 mg cm⁻².

3.5. Gas Exchange Parameters

Seven treatments were selected to determine gas exchange parameters (Figure 6). Leaf net photosynthesis (P_n) strongly declined with Mg scarcity both in rice and cucumber seedlings. The critical Mg supply concentrations for P_n of rice and cucumber leaves were 0.02 and 0.1 mM, respectively. With the 0.1 mM Mg treatment as control, the P_n of the 0.02 mM Mg treatment significantly declined by 36.7% in cucumber; however, no significant change was found in rice. The decrease in Mg supply significantly reduces g_s and T_r, while C_i was not significantly affected by differential Mg treatments.

3.6. The Critical Mg Threshold for Biomass, Chl a+b, P_n, and LA

To compare the differences in the response of different indicators to Mg, we fitted the relationships of total biomass, Chl a+b, P_n, and LA with leaf Mg concentration using a linear-plateau model, and found the corresponding critical Mg thresholds (Figure 7; Table S2, Supplementary Materials). From Figure 4, we know that Mg affects chlorophyll a and b more than carotenoids, so we compare here the Mg threshold for Chl a+b. Critical leaf Mg concentration range values for Chl a+b at different leaf positions in rice and cucumber were 0.98–1.22 and 2.88–4.23 mg g⁻¹ DM, respectively. The lowest value occurred in the middle and lower leaves, which displayed apparent Mg-deficiency symptoms in both plants. The Mg thresholds value for Chl a+b was 1.22 and 4.23 for rice and cucumber, respectively, for leaf positions corresponding to other indicators. We found a critical leaf Mg threshold (0.97 mg g⁻¹ DM) corresponding to rice biomass; however, cucumber biomass did not respond well to changes in leaf Mg concentration. The critical leaf Mg concentration was 1.05 mg g⁻¹ DM for P_n and 1.00 mg g⁻¹ DM for LA in rice, which was lower than that of 4.09 mg g⁻¹ DM and 3.55 mg g⁻¹ DM in cucumber (Figure 7). The critical leaf Mg concentrations for growth indicators (e.g., total biomass and LA) is lower than that for physiological indicators (e.g., Chl a+b and P_n). In terms of chlorophyll, photosynthetic rate, and leaf area, cucumber corresponds to a higher Mg threshold than rice.

4. Discussion

4.1. Critical Mg Thresholds for Chlorophyll Were Higher Than Those for Photosynthesis, Leaf Area, and Biomass, Suggesting That Chlorophyll Is More Sensitive to Mg Deficiency Stress

Although numerous studies addressed the physiologic and phenotypic responses of plants to Mg deficiency [15,16,17] and reported critical Mg concentration that are commonly used for plant dry weight or yield, it is not yet elucidated whether this value also applies to other physiological processes. In our study, the linear-with-plateau model illustrated the relationships among biomass, chlorophyll, leaf area, photosynthetic rate, and leaf Mg concentration. We found a leaf Mg threshold of 1.22, 1.05, and 1.00 mg g⁻¹ DM for Chl a+b, P_n, and LA, respectively, and a threshold of 0.97 mg Mg g⁻¹ DM for total biomass in rice seedlings (Figure 7). The results showed that the leaf Mg threshold for biomass was close to that for Pn and LA, and slightly lower than that for chlorophyll in rice plants. For comparison purposes, we also found a plant tissue Mg threshold of 1.46 mg g⁻¹ DM for total biomass (Figure S3, Supplementary Materials). It is consistent with the reported value of 1.50 mg g⁻¹ DM for biomass and yield formation [1,19]. In cucumber seedlings, Mg deficiency significantly reduced biomass when Mg supply concentration was below 0.05 mM; however, no Mg threshold value was found (Figure 1 and Figure 7). The values of the Mg threshold for Chl a+b, P_n, and LA in cucumber were 4.23, 4.09, and 3.55, respectively. These results suggest that, compared to biomass, photosynthesis, and leaf area, chlorophyll is more sensitive to Mg deficiency stress in both crops and can be used as an early diagnostic indicator of Mg nutrition. In addition, the order of sensitivity of the four indicators to Mg was chlorophyll > photosynthesis > leaf area > biomass. This is consistent with research on tomatoes that the critical leaf Mg concentrations for each indicator showed SPAD (4.67, 5.52) > photosynthesis (4.41, 5.01) > plant DM (4.38, 4.50) in both the first and second harvests of tomato [37]. Hauer-Jákli and Tränkner [10] reported that the calculated and cited critical levels for net CO₂ assimilation were mostly higher than for biomass or harvestable yield in rice, cotton, sugar beet, coffee, and potatoes, as also described by [23]. All these findings are consistent with the results of rice and cucumber in this study, implying that physiologically important processes for plant growth can be already negatively affected, although Mg supply is not limiting biomass. Moreover, it implies that photosynthetic rate is not the only prerequisite for maximal dry matter or yield. Therefore, critical Mg levels for chlorophyll rather than for LA, P_n, and crop growth parameters should be used to define the plant Mg status, since it allows early recognition of Mg stress before biomass formation is affected.

4.2. The Upper Young Mature Leaves Had a Higher Chlorophyll Critical Mg Threshold, Whereas Visible Symptoms of Mg Deficiency Were Predominantly Found in Mid-Aged Leaves with a Higher Rate of Mg Remobilization

Owing to Mg occupying the key position in chlorophyll a and b, interveinal chlorosis of Mg-deficient leaves is closely related to the degradation of chlorophyll [15,38,39]. Although the visual symptoms are mainly observed in leaves at some leaf positions, Chl a, Chl b, and Car at all leaf positions/ages significantly decreased with the reduction in leaf Mg in both seedlings (Figure 4). This differs from studies on Citrus sinensis seedlings that reported that Mg-deficiency-induced alterations of pigments were observed in middle and lower leaves but not in upper leaves [25,26]. This may be related to the critical Mg thresholds corresponding to chlorophyll at different leaf positions. In this study, critical Mg thresholds for all chlorophyll components were higher in the upper leaves (e.g., L1 in rice and upper in cucumber) of both crops compared to other leaf positions, showing a tendency for the values to decrease with increasing leaf age (Figure 4). It is worth noting that Chls and leaf Mg were linearly negatively correlated, and no critical Mg threshold was found for the lower cucumber leaves. These results suggest that upper functional leaves, which have a more urgent demand for Mg, are more sensitive to Mg deficiency and more susceptible to Mg stress affecting intrinsic physiological processes. Under the conditions of this experiment, the Mg concentration of 1.19–1.33 mg g⁻¹ DM in rice L1 leaves and 4.19–4.28 mg g⁻¹ DM in cucumber upper leaves can be considered as their respective Chl critical Mg thresholds. The Car/Chl a+b ratios were significantly negatively correlated with leaf Mg concentration in both seedlings, indicating that Chl a+b declined more rapidly than Car with decreasing Mg concentration. Chl a+b was more sensitive to Mg deficiency than Car, and it can be used as a more precise diagnostic indicator when making magnesium nutritional diagnoses.

It is generally accepted that the leaf age/positions that are more sensitive to Mg may show more pronounced symptoms of Mg deficiency. However, visual Mg-deficiency symptoms occurred preferentially in middle and lower leaves. For example, the inter-venial chlorosis symptoms in leaves was more visible in cucumber, especially in middle mature leaves (Figure 1). This indicates a prioritization in reutilizing Mg within the middle leaf positions. Similar trends were observed in sugar beet [15], Arabidopsis [40,41], and rice [42,43]. It reinforced the previous study that Mg retranslocation from old mature leaves is not as vigorous as that from young mature leaves [42]. When exposed to low Mg stress, plants preferentially reutilize Mg of the mid-aged mature leaves [43,44], which was confirmed by the faster and more rapid decline in Mg level in the middle of cucumber than in upper and lower leaves in our study (Figure 3). At the whole-leaf level, the withdrawal of Mg in cucumber appeared to occur initially at the center of the leaf with vigorous metabolism (Figures S1 and S2, Supplementary Materials). That aligns with the findings of Peng et al. [43] that upregulation of OsSGR (STAY-GREEN) is an adaptive strategy to accelerate Mg remobilization and protect mid-aged leaves from photodamage under low Mg stress. In addition, as the supply Mg concentration decreased, the proportion of decrease in Mg concentration at different leaf positions was ranked as Rest > L3 > L2 > L1 leaves in rice and middle > upper > lower in cucumber, suggesting that Mg concentration at different leaf position responds differently to Mg deficiency (Figure 3). Differences in leaf Mg concentrations under Mg deficiency due to the leaf age were observed across various plants, e.g., Brassica napus [44], rice [30], Citrus [25], and barley [39]. Therefore, the Mg content of mid-aged leaves itself is relatively lower than other ages of leaves [15,30,43], and these leaves easily fall below the general threshold value in leaves for the occurrence of deficiency symptoms, reported to be about 2 mg g⁻¹ DM [45]. Meanwhile, within an individual leaf of cucumber, Mg concentration was remarkably higher in the margins than in centers, which was an opposite trend to the changes in chlorophyll, and the gap increased with increasing Mg supply (Figure S2, Supplementary Materials). This further illustrates the importance of blade position. Above all, leaf position should be taken into account when making a morphological or chemical diagnosis of Mg nutrition to prevent and alleviate issues arising from Mg deficiency early.

4.3. In Terms of Chl, LA, P_n, and Biomass, Cucumber Was More Sensitive to Mg Deficiency Stress Compared to Rice

A broad survey of the literature reveals that a higher demand for Mg is required for vegetables than for cereal, mostly focused on the total amount of Mg fertilizer/rate for the optimal plant biomass and/or yield, ignoring the different sensitivity of physiological processes for plant growth to Mg [10,33]. In the present study, we compared the difference in growth phenotypes for rice and cucumber seedlings under a series of Mg levels and identified the corresponding Mg supply threshold values. Results showed that the Mg supply threshold values for phenotypes of rice and cucumber seedlings were 0.02 and 0.2 mM (Figure 1), respectively, suggesting that the growth of cucumber seedlings is more sensitive to low Mg stress than that of rice seedlings. The differences might partly account for the diverse responses of dicots and monocots to Mg deficiency. The symptoms of Mg mainly exhibit severely stunted growth in rice seedlings and inter-venial chlorosis of leaves in cucumber. Our study also showed that chlorophyll pigments, leaf area, and photosynthetic capacity of cucumber (dicots) reacted more sensitive to Mg deficiency than that of rice (monocots, Figure 4 and Figure 7). This is consistent with previous studies that showed that dicots reacted with more sensitivity than monocots to decreasing Mg concentrations [10,22]. When comparing the correlations among the Chl a, Chl b, Car, Car/Chl a+b ratio, and Mg concentration, rice exhibited a more rapid response to Mg deficiency than cucumber (Figure 4). In considering the role of Car in scavenging ROS, cucumber was equipped with lower Car under Mg deficiency and may have suffered more oxidative stress than rice. Meng et al. [34] also showed that cucumber was more sensitive to Mg deficiency than rice due to lower NPQ, higher rates of electron transport to alternative pathways, and, subsequently, photooxidation damage. These differences might be due to varying physiological utilization and stage-specific requirements of Mg between dicots and monocots. Given the inconsistent sensitivity of different crops to the effects of Mg deficiency, crop types should be taken into account when performing crop Mg nutrition diagnosis. In our study, the chlorophyll content of L1 leaves in rice and upper leaves in cucumber exhibits a greater sensitivity response to Mg deprivation compared to photosynthetic rate and leaf area. Therefore, we can use the chlorophyll content, especially Chl a+b, as a key indicator to assess the Mg nutritional status of crops, as it can be assessed even before apparent Mg deficiency symptoms manifest. When performing Mg nutrition analysis, crops that are more sensitive to magnesium deficiency can be judged on the basis of leaf deficiency symptoms, while crops that are not sensitive to magnesium deficiency can be measured for intrinsic physiological indicators.

Some studies emphasized that determining critical Mg concentration should consider variety, growth stage, the growing condition, and the analyzed tissue’s age [29,46]. Moreover, Bielczynski et al. [47] reported that photosynthetic performance was shown to be affected by leaf age and growth stage in Arabidopsis. However, Hauer-Jákli and Tränkner [10] suggested that the leaf age, growing condition, or plant growing stage might be of minor relevance for the critical Mg thresholds of net CO₂ assimilation as the photosynthetic performance of a leaf primary depends on Mg tissue concentration (assuming other stresses or nutrient deficiencies can be excluded). In the present study, all measurements were sampled at the seedling stage and also took into account leaf age to eliminate the differences caused by these factors. Here, twelve different Mg supply levels were chosen to induce a gradient in Mg tissue concentrations. Determining the critical Mg threshold has a certain limitation under greenhouse conditions in our study, since multiple biotic and abiotic stress factors are likely to occur in the field during biomass formation. In brief, our results imply the importance of considering species-specific responses to Mg deficiency, offering insights into potential differences in nutrient requirements and responses among different crops, which can guide more precise and effective nutrient management strategies in agriculture.

5. Conclusions

The present study showed that reduction in external Mg supply to a certain value significantly decreased biomass, Chl, P_n, and LA and increased SLM in rice and cucumber. The critical leaf Mg concentrations were found to be 1.22 mg g⁻¹ DM for Chl a+b, 1.05 mg g⁻¹ DM for P_n, and 1.00 mg g⁻¹ DM for LA in rice seedlings, which were lower than those of 4.23, 4.09, and 3.55 mg g⁻¹ DM in cucumber, respectively. The findings indicate that various physiological indicators exhibit distinct sensitivities to Mg deficiency, with each showing a decline once the leaf Mg concentration falls below its critical threshold. The main findings were that Chl is the most sensitive to low Mg stress compared to P_n and LA in both plants; cucumber was more susceptible to Mg-deficiency stress compared to rice assessed with the chlorosis level, Chls, P_n, and LA. Additionally, the critical Mg threshold for chlorophyll varies according to leaf position/age and chlorophyll type. Leaf Mg critical thresholds for Chls tended to decrease with leaf age in both crops, suggesting Chls in the upper young mature leaves was more sensitive than that in other leaves to Mg starvation. However, the visible symptoms of Mg deficiency were predominantly found in cucumber mid-aged leaves due to the higher rate of Mg remobilization. Regardless of the leaf position, Chl a and b respond preferentially over carotenoids to the reduction in Mg supply. Nevertheless, rice biomass was more affected by Mg deprivation compared to cucumber, exhibiting severely stunted growth, and the critical Mg value for rice seedlings was 0.97 mg g⁻¹ DM, which does not necessarily apply to other physiological indicators. Overall, these results underscore the necessity for leaf position/age, determination of physiology parameters, and crop species in making Mg nutritional diagnosis. Chlorophylls in upper young mature leaves can be used for the early detection of Mg deficiency, especially for Mg-insensitive crops that are not prone to visual symptoms such as rice. Taken together, these findings provide insight into the differential sensitivity of dicots and monocots to Mg deficiency, highlighting variations in their physiological responses to this stressor, and increase our knowledge for specific Mg nutrient management of various crops.