Supplemental Foliar-Applied Magnesium Reverted Photosynthetic Inhibition and Improved Biomass Partitioning in Magnesium-Deficient Banana

Abstract

Magnesium (Mg) is an essential macronutrient in plants and plays a critical role in numerous physiological processes. Therefore, Mg deficiency severely affects plant growth and crop production. This study aimed to investigate the effects of Mg deficiency on plant growth, biomass formation, Mg homeostasis, and photosynthesis of banana seedlings. After exhibiting deficiency symptoms, plants were sprayed with Mg to alleviate the deficiency. Mg deficiency severely reduced plant biomass and chlorophyll content. A significant reduction in maximum quantum yield (F_v/F_m), the effective quantum yield of PS II (Φ_PSII), photochemical fluorescence quenching (qP), and non-photochemical fluorescence quenching (NPQ) was observed. In contrast, the light compensation point was almost doubled under Mg deficiency. This indicated damage to the photosynthetic apparatus and photoinhibition under Mg-deficiency treatment. The foliar application of Mg to Mg-deficient plants significantly increased the biomass and reversed the decrease in the biomass of leaves, pseudostem, and corms. More improvement was observed in the leaf area and biomass of the upper leaves. Foliar Mg also increased the Mg concentration in all tissues and enhanced chlorophyll content and chlorophyll fluorescence in leaves. In conclusion, foliar Mg application to Mg-deficient plants efficiently restored banana plant development and might be a practical approach to correcting Mg deficiency in the field.

1. Introduction

Bananas (Musa spp.) are a major fruit crop in the tropics and subtropics, providing a vital contribution to the economy in many countries [1]. It is well-known that the banana fruit is a natural potassium (K) source and requires a high concentration of nutrients due to its fast growth rate; therefore, in routine agricultural practice, the plants are supplied with a surplus of N, P, and K fertilizers [2]. Many commercial banana plantations are highly prone to magnesium (Mg) deficiency due to the over-application of N and K fertilizers, less Mg supplementation, and an intensive cropping system [3]. High soil K and NH₄⁺ levels strongly inhibit Mg uptake from the soil and have an antagonistic effect on Mg uptake and transport in the plant cell’s metabolic pool [4,5].

Most of the Mg in the soil is bound in the crystal lattice structure of minerals, and 90–98% of it cannot be absorbed directly by plants [6]. The only form that plants can absorb is Mg²⁺, but this form is highly prone to leaching in acidic soil, because it is weakly bound to root cell walls and negatively charged soil particles due to its large size [7]. Therefore, Mg deficiency frequently occurs in acidic and light-textured soils with low cation exchange capacities, particularly in areas with heavy rainfall. The competing cations, e.g., H⁺, Al³⁺, K⁺, Ca²⁺, Na⁺, and NH₄⁺, displace Mg²⁺ from the cation exchange sites; Mg²⁺ leaches, and plant roots cannot uptake Mg²⁺ [8].

Mg is involved in various physiological and biochemical processes. For example, Mg²⁺, a central atom of the chlorophyll molecule in the light-absorbing complex of chloroplasts, plays a vital role in photosynthesis [9,10]. In addition, Mg is a cofactor and allosteric modulator for more than 300 classes of enzymes, including many photosynthetic enzymes in chloroplasts involved in carbon fixation, ATPases, RNA polymerases, phosphatases, kinases, and carboxylases [11].

Mg deficiency is known to trigger obvious chlorosis in the leaf. The first visual symptoms of Mg deficiency are usually observed in older leaves with typical interveinal chlorosis and an inverted V-shaped green area at the leaf base [4,12,13]. Because of the disturbed physiological status, Mg deficiency reduces biomass accumulation, yield, and quality [14,15]. Mg deficiency causes an imbalance in carbohydrate transport between source and sink organs [16,17]. The carbohydrate is accumulated in the source leaf by plasma membrane-bound ATPases, resulting in the inhibition of photosynthate export via the phloem [18,19]. In addition, carbohydrate accumulation inhibits photosynthesis via down-regulation [20].

It is mostly assumed that Mg deficiency reduces net photosynthesis due to non-stomatal factors [21,22] rather than affecting stomatal conductance [10]. Unused electrons accumulate in the chloroplast due to impaired photosynthesis, causing the production of reactive oxygen species (ROS), and consequently, ROS damages the chloroplast and photosystem II (PS II) [10].

The pattern of photosynthetic response varies with plant species and the intensity and duration of Mg deficiency. For instance, complete deprivation of Mg did not change the photosynthetic parameters of the sunflower after two weeks [23] and in Eustoma after eight weeks of Mg deficiency [24]. In Mg-deprived maize, about a 20% reduction in electron transport in the photosystem I (PS I) was detected [25], and in that of rapeseed and buckwheat, PS I photosynthetic activity even increased [26]. In pine seedlings, a decline in photosynthetic CO₂ fixation rate and gs was observed, but PS II photochemistry (F_v/F_m) was unchanged [27]. Though both PS I and PS II were affected, photosynthetic electron transport was impaired in citrus [28] and sugar beet [29].

Basal and foliar Mg applications effectively alleviate Mg deficiency [13]. However, the effectiveness of root application is influenced by various environmental factors, such as dry soil and low transpiration rates. Foliar Mg application might be more advantageous than Mg fertilization in soil, mainly when the physiological demand for Mg is high. The positive effects of Mg foliar application on plant growth have already been described for some crops such as wheat [30], maize [13], soybean [31], and faba bean [14]. However, plant responses to foliar application differ greatly among plant species due to diverse leaf structural properties such as cell wall composition, cuticle chemistry, and trichome formation [32].

Although the nutrient allocation and biomass partitioning after Mg deficiency have been analyzed in our previous work [17], the photosynthetic response of bananas to Mg deficiency and the alleviation of the effect by foliar Mg application is still unknown. We hypothesized that foliar Mg application could effectively reverse the adverse impact of Mg deficiency on photosynthetic traits by increasing tissue Mg concentrations in banana seedlings. The aim of this study is, therefore, to examine the detailed effect of Mg deficiency and foliar Mg application on the growth and photosynthesis of banana seedlings. Furthermore, the results of this study could provide guidance for Mg fertilization in Mg-deficient soil.

2. Materials and Methods

2.1. Seedling Culture and Mg Treatments

A pot experiment was conducted in a greenhouse from 7 July to 29 September 2018, under natural temperature and photoperiod at Hainan University, Haikou, China (20.0592° N, 110.3247° E). Seedlings of the banana (Musa sp. AAA group, cv. Baxijiao, Cavendish subgroup) were derived from tissue-cultured plantlets. Uniform seedlings with four fully expanded leaves were transferred into 7-L plastic pots containing cleaned quartz sand (one plant per pot). Three treatments were used, including twelve weeks of sufficient Mg supply (control treatment, +Mg), twelve weeks of deficient Mg supply (−Mg), and six weeks of foliar Mg application after Mg-deficiency treatment for six weeks of (−Mg/+Mg). At least 10 seedlings (one seedling per pot) were used in each treatment. A total of 36 pots were used in the experiments, and they were randomly arranged. For growth and recovery during the first week, only deionized water was supplied to the seedlings. In the second and third weeks, the seedlings were successively provided with 1/4, 1/3, 1/2, and full-strength Hoagland nutrient solutions containing either 1 mM Mg (+Mg) or 0 mM Mg (−Mg)(−Mg and −Mg/+Mg) every two days. The full-strength modified Hoagland nutrient solution [33] contained 6 mM KNO₃, 4 mM Ca(NO₃)·4H₂O, 2 mM NH₄H₂PO₄, 4 mM KCl, 1 mM MgSO₄·7H₂O, 60 µM Fe-EDTA, 25 µM H₃BO₃, 2 µM MnSO₄·H₂O, 2 µM ZnSO₄·7H₂O, 0.5 µM CuSO₄·5H₂O, and 0.05 µM H₂MoO₄. Every time, 500 mL of nutrient solution was supplied to each seedling. Six weeks after transplanting, the tissues were sampled from +Mg and −Mg treatments. Then, the total plants were divided into two parts, and the first half of the Mg-deficient seedlings were foliar-sprayed with 2% (w/v) MgSO₄·7H₂O solution (−Mg/+Mg) mixed with 0.01% Tween-20, whereas the other half of seedlings (−Mg) were sprayed with deionized water. The foliar application was conducted every 3.5 days, four times in two weeks. All the leaves were thoroughly sprayed on both sides until water droplets formed. Twelve weeks after transplanting, the second sampling was conducted for all three treatments. All treatments were repeated at least three times.

2.2. Sampling and Measurements

At 6 and 12 weeks of transplantation, three pots were randomly selected from each treatment. Banana plantlets were carefully taken out of quartz sand, washed using deionized water, and separated into leaves, pseudostems, rhizomes, and roots. The first new fully expanded leaf was regarded as the upper leaf, the 2nd to the 4th leaves were regarded as middle leaves, and the remaining leaves below were regarded as lower leaves. Another three plantlets were sampled from each treatment. Four to six leaves from the bottom of the three plantlets were collected, frozen in liquid nitrogen, and stored at −80 °C before pigment measurement.

2.3. Mg Determination

All the samples were oven-dried to a constant weight to determine their Mg content. About 0.5 g of powder per sample was digested using HNO₃–HClO₄–HCl, and Mg content was determined by the atomic absorption spectrophotometer (AAnalyst 400, Perkin Elmer cooperation) [34].

2.4. Leaf Chlorophyll Content

All leaf samples were ground in liquid nitrogen. About 0.2 g samples were used to extract chlorophyll with 10 mL 95% ethanol at 4 °C for 24 h in the dark before measuring the absorbance. The chlorophyll content was calculated according to the method described by Wellburn and Lichtenthaler [35].

2.5. Leaf Gas Exchange Measurement

The second fully expanded young leaf from the top of the plantlets, which is considered to be the source leaf, was used to determine the net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO₂ concentration (C_i), and transpiration rate (E) using a portable gas exchange system (Li-6400, LI-COR Biosciences, Lincoln, NE, USA). Measurements were taken at 9:00–11:00 a.m. on a sunny day with leaf temperatures of 27 °C and a relative humidity of 60%. Incoming photosynthetic photon flux density was provided by a red/blue LED light source (1200 µmol m⁻² s⁻¹), and the ambient CO₂ concentration was adjusted to 400 µmol mol⁻¹ via CO₂ injection.

The light response curve was measured using the same leaf as described above under constant leaf temperature (27 °C), leaf humidity (60%), and CO₂ concentration (400 µmol mol⁻¹). The leaf was placed under 600 µmol m⁻² s⁻¹ for 20 min to activate the photosynthesis systems fully. The light series of photosynthetic light flux densities were set as 2600, 2300, 2000, 1700, 1400, 1000, 800, 600, 400, 200, 150, 100, 50, 20, and 0 µmol m⁻² s⁻¹. The responses of net photosynthetic rate to different lights were modeled using the non-rectangular hyperbola in the Farquhar model [36]. The quantum yield was estimated from the initial slope by fitting a linear regression to the low-photon flux data (less than 200 µmol m⁻¹) of the light response curve. The point at which the curve interacts with the X-axis is referred to as the LCP (the light compensation point, µmol m⁻¹). The Amax is derived from the Y value of the curve where the X-axis corresponds to the LSP (the light saturation point).

The response of light-saturated CO₂ assimilation to variable internal CO₂ concentrations (A-C_i curves) was measured under constant leaf temperature (27 °C), leaf humidity (60%), and photosynthesis saturating irradiance of 1200 µmol m⁻² s⁻¹ using the same leaf. The CO₂ concentrations series were set as 0, 25, 50, 80, 100, 150, 200, 350, 500, 800, 1000, 1200, 1400, 1600, 1800, and 2000 µmol mol⁻¹. A Pn response curve was also made using the above-described conditions. CO₂ compensation point (CCP) and CO₂ saturation point (CSP) could be calculated. Data with Ci less than 200 µmol m⁻² s⁻¹ were used for linear regression; the straight slope was carboxylation efficiency (CE).

The Pn-PAR curve and the Pn-CO₂ curve were formulated using a non-rectangular hyperbolic model for simulation [37], in which the light-saturated photosynthetic rate (A_maxl, µmol CO₂ m⁻² s⁻¹) and the CO₂ saturated photosynthetic rate (A_maxc, µmol CO₂ m⁻² s⁻¹) could be calculated. The parameter apparent quantum yield (AQY) as assessed by the following function:

$$\mathrm{Pn}=\frac{\mathrm{aI}+{\mathrm{A}}_{\max }-\sqrt{{\left(\mathrm{aI}+{\mathrm{A}}_{\max }\right)}^2-4{\mathsf{\theta }\mathrm{aIA}}_{\max }}}{2\mathsf{\theta }}-{\mathrm{R}}_{\mathrm{d}}$$

(1)

where Pn is the net photosynthetic rate, $\mathrm{a}$ is apparent quantum yield, $\mathrm{I}$ is the photosynthetic active radiation (PAR) or CO₂ concentration, ${\mathrm{A}}_{\max }$ is the maximum net photosynthetic rate, $\mathsf{\theta }$ is the curvature of the light response curve, and R_d is the dark respiration rate.

2.6. Chlorophyll Fluorescence Analysis

All chlorophyll fluorescence parameters were taken with a Monitoring-PAM Multi-Channel Chlorophyll Fluorometer (Walz, Effeltrich, Germany) from the same leaf chosen for gas exchange measurement. Measurements were taken from 9:00 to 11:00 am to avoid the errors caused by the diurnal changes in chlorophyll fluorescence [37].

Measurements were taken from each treatment. First, plants were placed in the dark for 15 min. Then, the minimum fluorescence yield of dark-adapted leaves (F₀) was measured under a measuring light (photosynthetic active radiation (PAR) of less than 2 μmol photons m⁻² s⁻¹). The maximum fluorescence yield of dark-adapted leaves (F_m) was measured after 0.6 s’ saturating pulse light. The maximum fluorescence yield of light-adapted leaves (F’_m) was measured after 8 cycles of 30 s of actinic light (125 μmol photons m⁻² s⁻¹ ), followed by 0.6 s of saturating pulse light. The maximum quantum yield of PS II (F_v/F_m) and the effective quantum yield of PS II (Φ_PSII) were calculated following the equations below [38]:

F_v/F_m= (F_m − F₀)/F_m

(2)

F_v/F₀= (F_m − F₀)/F₀

(3)

Φ_PSII = (F′_m − F)/F′_m

(4)

$$\mathrm{qP}=\frac{\left({\mathrm{F}\prime }_{\mathrm{m}}-\mathrm{F}\right)}{\left({\mathrm{F}\prime }_{\mathrm{m}}-{\mathrm{F}}_0\right)}$$

(5)

$$\mathrm{NPQ}=\frac{{\mathrm{F}}_{\mathrm{m}}}{{\mathrm{F}\prime }_{\mathrm{m}}}-1$$

(6)

$$\mathrm{Y}\left(\mathrm{NPQ}\right)=\frac{\mathrm{F}}{{\mathrm{F}\prime }_{\mathrm{m}}}-\frac{\mathrm{F}}{{\mathrm{F}}_{\mathrm{m}}}$$

(7)

$$\mathrm{Y}\left(\mathrm{NO}\right)=\frac{\mathrm{F}}{{\mathrm{F}}_{\mathrm{m}}}$$

(8)

2.7. Root Morphology and Root Dry Weight

The cleared root samples were dispersed with water in a transparent box (30 cm × 20 cm × 2 cm) and scanned using an Epson Expression 1680 scanner (Seiko Epson, Nagano, Japan) at 800 dpi. The images were analyzed by WinRhizo v. 2009c software (Regent Instrument Inc., Quebec QC, Canada) to determine the total root length and root surface area. After scanning, the root samples were used to measure total root dry weight.

2.8. Statistical Analysis

After Mg deficiency for six weeks, all the data were subjected to a paired sample t-test between +Mg and −Mg treatment (SPSS20.0, New York, NY, USA); after 12 weeks of treatments, all the data were subjected to one-way analysis of variance (ANOVA), and the differences between treatments were compared using the t-test.

3. Results

3.1. Growth and Biomass Partitioning

3.1.1. Mg Deficiency Reduced the Growth and Altered the Biomass Partitioning of Banana Seedlings

The banana seedlings supplied with 1 mM Mg (+Mg) showed normal growth without any visual chlorosis, whereas six weeks of Mg deficiency (−Mg) caused chlorosis at the edges of the second leaf from the top (Figure 1). Mg deficiency significantly decreased the Mg concentration in source leaves (Figure 2). The leaf area of the newly developed leaves was significantly reduced after 6 weeks’ Mg deficiency; after 12 weeks, the leaf area was significantly decreased in all leaves (Figure 3).

Mg deficiency significantly inhibited the growth of banana seedlings (

Table 1. Mg deficiency and foliar Mg application affected the growth of banana seedlings.

	Treatment	Plant Height (cm)	Shoot Diameter (mm)	Leaf Number	Root Diameter (mm)	Total Root Length (cm)	Root Surface Area (cm2)	Root Volume (cm3)
6 weeks	+Mg	19.82 ± 1.02 c	18.52 ± 1.68 b	10.14 ± 0.38 a	0.67 ± 0.02 a	84.87 ± 23.87 b	1705.22 ± 430.57 c	29.90 ± 6.76 b
	−Mg	17.10 ± 1.34 d	18.12 ± 1.36 b	9.67 ± 0.52 a	0.60 ± 0.01 b	53.78 ± 21.34 b	1051.11 ± 432.48 c	16.11 ± 6.39 c
12 weeks	+Mg	31.21 ± 1.70 a	30.30 ± 1.50 a	10.25 ± 0.50 a	0.62 ± 0.07 b	266.07 ± 46.98 a	3987.60 ± 603.08 a	48.57 ± 8.51 a
	−Mg	28.67 ± 1.40 b	31.56 ± 4.12 a	8.20 ± 1.30 b	0.49 ± 0.05 c	59.62 ± 23.86 b	1171.74 ± 486.75 c	17.91 ± 5.72 c
	−Mg/+Mg	30.75 ± 1.44 a	31.13 ± 1.00 a	9.75 ± 1.71 a	0.61 ± 0.02 b	95.57 ± 29.65 b	1892.31 ± 465.26 b	28.61 ± 7.72 b

+Mg, 1 mM Mg in nutrition solution; −Mg, 0 mM Mg in nutrient solution; −Mg/+Mg, 0 mM Mg in nutrition for 6 weeks and foliar application of 2% (w/v) MgSO₄·7H₂O four times. Data are presented as mean ± standard error (n = 3). Data with different letters are significantly different between treatments at p < 0.05 level (t-test).

). After six weeks of Mg deficiency, about a 4.6% reduction in leaf number was observed, reaching 20% after 12 weeks as compared to Mg-sufficient control plantlets. The underground part of the seedling was more severely affected by Mg deficiency. The root length, root surface area, and root volume decreased by more than 35% after six weeks and 60% after 12 weeks of Mg deficiency.

Mg deficiency significantly reduced banana dry matter production (

Table 2. Mg deficiency and foliar Mg application altered the dry matter partitioning in banana seedlings.

	Treatment	Leaf Position			Pseudostem (g)	Corm (g)	Root (g)	Aboveground (g)	Whole Plant (g)	Root DW/Shoot DW
		Upper Leaf (g)	Middle Leaves (g)	Lower Leaves (g)
6 weeks	+Mg	1.12 ± 0.15 b	2.90 ± 0.22 c	1.57 ± 0.10 d	3.26 ± 0.09 c	1.63 ± 0.06 c	2.31 ± 0.26 c	8.86 ± 0.46 d	12.80 ± 0.65 d	0.22 ± 0.02 b
	−Mg	0.73 ± 0.08 c	2.12 ± 0.18 d	1.37 ± 0.03 e	2.79 ± 0.19 c	0.99 ± 0.09 d	1.05 ± 0.11 d	7.11 ± 0.35 e	9.14 ± 0.47 e	0.13 ± 0.01 c
12 weeks	+Mg	2.12 ± 0.15 a	3.93 ± 0.39 a	5.31 ± 0.12 a	6.20 ± 0.25 a	3.78 ± 0.27 a	6.09 ± 0.63 a	17.56 ± 0.39 a	27.43 ± 0.81 a	0.29 ± 0.03 a
	−Mg	1.04 ± 0.24 b	1.83 ± 0.09 e	4.65 ± 0.02 c	5.16 ± 0.49 b	2.07 ± 0.16 b	1.65 ± 0.33 c	12.68 ± 0.69 c	16.40 ± 0.91 c	0.11 ± 0.02 c
	−Mg/+Mg	2.16 ± 0.23 a	3.24 ± 0.33 b	4.83 ± 0.11 b	6.37 ± 0.31 a	3.60 ± 0.12 a	5.03 ± 0.54 b	16.61 ± 0.35 b	25.24 ± 0.76 b	0.25 ± 0.03 a

+Mg, 1 mM Mg in nutrition solution; −Mg, 0 mM Mg in nutrient solution; −Mg/+Mg, 0 mM Mg in nutrition for 6 weeks and foliar application of 2% (w/v) MgSO₄·7H₂O four times. Data are presented as mean ± standard error (n = 3). Data with different letters are significantly different between treatments at p < 0.05 level (t-test).

). After six weeks, the leaf dry weight of Mg-deficient plantlets was significantly lower than that of the Mg-normal control. The 12 weeks of Mg deficiency induced a more significant reduction in the dry weight of the upper and middle leaves. The dry weight of the middle leaves and roots was reduced to 26.8% and 54.5% after six weeks and 53.4% and 72.9%, respectively, after 12 weeks of Mg deficiency compared to the Mg-normal control. The 6 weeks of Mg deficiency significantly reduced the dry weight of the pseudostem, corm, and root by 14.4, 39.2, and 54%, respectively, as compared to the Mg-sufficient control plantlets. As a result, the plant root–shoot dry weight ratio decreased from 0.22 in the Mg-normal control to 0.11 in the Mg-deficient plantlets after 12 weeks.

3.1.2. Foliar Application of Mg Recovered the Growth and the Biomass Partitioning of Banana Seedlings

The visual symptoms (chlorosis) of Mg deficiency ere relieved after six weeks of foliar application of 2% Mg (−Mg/+Mg) (Figure 1). Meanwhile, Mg concentrations in all tissues increased significantly as compared to Mg-deficient plants. After foliar Mg application, Mg content was higher than that of the Mg-sufficient control in leaves, but it was still lower in the rhizome and roots (Figure 2).

The foliar Mg application to Mg-deficient plantlets recovered plant height, leaf number, and root diameter to the same level as the Mg-sufficient plantlets. However, the total root length, root surface area, and root volume were only partially recovered after foliar application, which was significantly lower than that of the Mg-sufficient plantlets (Table 1).

After six weeks of the foliar application of Mg, the dry matter partitioning was partly recovered (Table 2). The dry matter of the upper leaf, pseudostem, and corm recovered almost completely. Dry matter of the middle leaves and the roots were increased with foliar Mg application, which was significantly higher than that of the Mg-deficient seedlings, although it is still significantly lower than that of the Mg-sufficient control. Again, the recovery extent of dry matter partitioning of underground biomass was far less than that aboveground after foliar Mg application.

The foliar Mg application significantly increased the leaf area of new leaves, similar to the Mg-normal plantlets. However, foliar Mg application to the Mg-deficient plantlets had no significant effect on the leaf area of the middle and lower leaves (Figure 3).

3.2. Leaf Pigments

3.2.1. Mg Deficiency Altered the Leaf Pigment Composition

Mg deficiency changed plant pigment composition (Figure 4). Chl a, Chl b, and Car in leaves were significantly decreased after Mg-deficiency treatment for six weeks. The treatment lasting for 12 weeks reduced Chl a, Chl b, and Car by 52%, 46%, and 62%, respectively, as compared with that of the Mg-sufficient control.

3.2.2. Foliar Mg Application Significantly Increased Pigment Content

The Chl a, Chl b, total Chl, and carotenoid content in leaves increased significantly after foliar Mg application; it reached similar levels as compared with the Mg-sufficient control plantlets. At the same time, the Chl a/b ratio and Car/Chl ratio decreased after foliar Mg application (Figure 4).

3.3. Photosynthesis and Photosynthetic Capacity of Banana Seedlings

3.3.1. Mg Deficiency Significantly Altered the Photosynthetic Capacity of Banana Seedlings

Photosynthesis of the second fully expanded leaves was analyzed (Figure 5). Mg deficiency significantly reduced Pn, gs, and E while increasing Ci. The water-use efficiency of the banana seedlings was not affected by the Mg deficiency.

The photosynthetic capacity of the banana seedlings was measured using A-Ci curves with the Farquhar–von Caemmerer–Berry (FvCB) model [39]. Mg deficiency for 12 weeks significantly altered the photosynthetic capacity of the banana seedlings (

Table 3. Mg deficiency and foliar Mg application changed the photosynthetic capacity of banana leaves.

Photosynthetic Parameters	Treatment
	+Mg	−Mg	−Mg/+Mg
Fv/Fm	0.82 ± 0.01 a	0.64 ± 0.05 b	0.79 ± 0.01 a
ΦPSII	0.46 ± 0.05 a	0.37 ± 0.02 b	0.46 ± 0.03 a
F0	254.74 ± 22.17 b	296.55 ± 10.53 a	209.35 ± 28.17 c
Fv/F0	4.08 ± 0.24 a	1.91 ± 0.33 c	3.45 ± 0.16 b
Fm/F0	4.98 ± 0.24 a	2.98 ± 0.33 c	4.37 ± 0.16 b
LSP (µmol m−2s−1)	793.13 ± 20.69 a	499.21 ± 12.44 b	677.50 ± 19.56 c
LCP(µmol m−2s−1)	33.94 ± 2.41 c	69.17 ± 3.02 a	49.48 ± 2.13 b
Amaxl (µmol CO2 m−2 s−1)	12.25 ± 1.21 a	5.11 ± 0.95 c	7.01 ± 0.83 b
Amaxc (µmol CO2 m−2 s−1)	20.69 ± 1.32 a	14.71 ± 1.59 c	17.90 ± 1.01 b
CSP (µmol)	1064.93 ± 12.43 c	1158.66 ± 9.54 a	1102.86 ± 8.60 b
CCP (µmol)	50.17 ± 1.03 c	77.62 ± 2.02 a	59.17 ± 1.85 b
AQY	0.03 ± 0.01 a	0.01 ± 0.00 c	0.02 ± 0.00 b
CE	0.04 ± 0.00 a	0.03 ± 0.00 b	0.03 ± 0.01 b

+Mg, 1 mM Mg in nutrition solution, −Mg, 0 mM Mg in nutrient solution, −Mg/+Mg, 0 mM Mg in nutrition for 6 weeks and foliar application of 2% (w/v) MgSO₄·7H₂O four times. Data are presented as the mean of three replicates. At 12 weeks, different letters in the same row indicate a significant difference between treatments at p < 0.05 level (t-test). Data in the bracket indicated the change percentage when compared with the corresponding control. F_v/F_m: Maximum photochemical efficiency of PS II, Φ_PSII: effective quantum yield of PS II, F₀: minimum fluorescence yield of dark-adapted leaves, F_v/F₀: potential activity of PS II, F_m/F₀: the electron transport through PS II, LSP: light saturation point, LCP: light compensation point, A_maxl: light-saturated photosynthetic rate, A_maxc: CO₂ saturated photosynthetic rate, CSP: CO₂ saturation point, CCP: CO₂ compensation point, AQY: apparent quantum yield, CE: carboxylation efficiency.

). Mg deficiency reduced the maximum quantum yield (F_v/F_m) from 0.82 of Mg-sufficient plants to 0.64 after 12 weeks of Mg deficiency. Similarly, the effective quantum yield of PSII (ΦPSII) was significantly reduced from 0.46 to 0.37. Mg deficiency significantly reduced F_v/F₀, F_m/F₀, light saturation point (LSP), light-saturated photosynthetic rate (Amaxl), CO₂-saturated photosynthetic rate (Amaxc), apparent quantum yield (AQY), and carboxylation efficiency (CE). In contrast, Fo, the light compensation point (LCP), the CO₂ saturation point (CSP), and the CO₂ compensation point (CCP) increased significantly after Mg deficiency.

Mg deficiency decreased the photochemical fluorescence quenching (qP), non-photochemical fluorescence quenching (NPQ), and light-induced non-photochemical fluorescence quenching [Y(NPQ)] (Figure 6A–C), but it increased the non-light-induced non-photochemical fluorescence quenching [Y(NO)] of the banana seedlings, especially under intensive light conditions (Figure 6D).

3.3.2. Foliar Mg Application Reversed the Adverse Effects of Mg Deficiency on Photosynthesis and Chlorophyll Fluorescence

The foliar Mg application to Mg-deficient plants increased the Pn, gs, and E and decreased Ci (Figure 5). The F_v/F_m and ΦPSII were recovered with foliar Mg application, which is similar to that of Mg-sufficient plants. The foliar Mg application enhanced F_v/F₀, F_m/F₀, LSP, Amaxl, Amaxc, AQY, and CE as compared to the Mg-deficient plants, but these traits were still significantly lower than that of the Mg-sufficient control. In contrast, Fo, LCP, CSP, and CCP showed reduction after foliar Mg application (Table 3).

The foliar Mg application significantly increased fluorescence quenching (qP), non-photochemical fluorescence quenching (NPQ), and light-induced non-photochemical fluorescence quenching [Y(NPQ)] (Figure 6A–C), while decreasing non-light-induced non-photochemical fluorescence quenching [Y(NO)] (Figure 6D).

4. Discussion

In the present study, the first symptom of Mg deficiency was observed at the edges of the second expanded leaf and then appeared on the new upper leaves, and necrotic spots developed on the older leaves. Similar Mg deficiency symptoms were previously reported by Chen and Fan [3] but were slightly different from other studies [7,22]. It has been proposed that the mobile characteristics of Mg induce phenotypic changes under Mg-deficient conditions [16,40,41]. The whole banana leaves were dissected into three parts, i.e., central, marginal, and midrib, and their Mg concentration was analyzed. After Mg deficiency for six weeks, more Mg concentration was detected at the marginal part compared with the center part and midrib, irrespective of the leaf position. In addition, Mg deficiency for six weeks significantly reduced Chl and Car concentration in the upper and middle leaves. Mg plays a role in protecting against photooxidative damage under high light conditions [7]. Thus, the abrupt reduction in Mg in the tissue might be responsible for pigment degradation and marginal chlorosis in the leaves.

Previous studies showed that 20 days of Mg deficiency caused chlorosis in the leaves [15,42,43]. However, symptoms appeared relatively late in banana seedlings and were observed after six weeks of Mg deficiency in a sand culture [3,17]. Thus, Mg deficiency exists long before the appearance of symptoms in banana plants. Mg deficiency reduced the Mg concentration in all banana tissues, such as the leaves, pseudostem, rhizome, and roots (Figure 1). Because of the critical functional role of Mg in numerous physiological processes, the lack of Mg eventually led to a reduction in plant growth and dry matter portioning [11,28,30]. The lower Mg concentration in the upper leaves and roots might harm the growth and development of the banana seedlings even after the restoration of tissue Mg concentration by foliar application.

Mg deficiency significantly reduced the growth and leaf area of bananas, especially the newly developed leaves. This might be because Mg deficiency induces vein lignification [44]. Mg deficiency affected the root growth more than that of the leaves, even though the first sign of deficiency symptoms was visible in the leaves. However, Mg deficiency induces a similar reduction in Mg content in leaves and roots. The length, surface area, and volume of the roots were reduced enormously, and the percentage was about 40% after six weeks and 70% after 12 weeks of Mg deficiency. In contrast, the reduction in shoots or leaves was less than 20%. Accordingly, a great discrepancy occurred in the dry matter partitioning of shoots and roots, resulting in a 40% and 60% reduction in root–shoot ratio after 6 and 12 weeks of Mg deficiency, respectively (Table 2). The activity of H⁺-ATPase, which is involved in sugar transport and active ion uptake by roots, requires the formation of the Mg-ATP complex [45]. The impaired root growth is a consequence of the impaired phloem export of photo-assimilates from source to sink organs that causes an increased shoot–root ratio under Mg deficiency [3]. Similar results were reported in beans, in which Mg deficiency for 12 d resulted in a 52% reduction in the root–shoot ratio [46]. A similar reduction was also observed in spinach [47], coffee plants [42], and Eustoma [24]. It has been well-observed that Mg deficiency induces alteration in gene expression and differential accumulation of metabolites, resulting in compromised growth [15,44]. Livigni et al. [15] reported differential accumulation of 771 and 791 transcripts in the roots of grapevine cultivars after 4 days of growth under Mg deficiency.

Some contradictory studies from our results showed that the Mg deficiency for 34 d caused no significant effect on barley’s root–shoot ratio [16] and Arabidopsis thaliania [48]. These results showed that plant species, genotypes, plant growth stage, duration of Mg deficiency, cellular Mg status, and the physiological demand of Mg in growth and development could contribute greatly to the dry matter partition after Mg deficiency [15,49]. The roots of banana seedlings are probably more prone to Mg deficiency and do not recover after foliar Mg. The foliar Mg application caused a significant increase in aboveground and root biomass, plant height, root diameter, and root surface area as compared to Mg-deficient plants. This might be because foliar Mg could reduce the oxidative stress in plants.

The effect of Mg deficiency on photosystem II activity was measured by examining F_v/F_m, F_v/F₀, Φ_PSII, qP, and NPQ. Our data showed that Mg deficiency significantly decreased F_v/F_m and F_v/F₀ simultaneously, and more reduction was observed in F_v/F₀. F_v/F_m reveals the maximum proportion of absorbed light used for photosynthesis. F_v/F₀ is a sensitive parameter and could be used to detect small changes in F_v/F_m [50,51，52]. According to Dan et al. [53], a 0.83 or higher value of F_v/F_m and a 4.0 or higher F_v/F₀ ratio indicate that the plant is healthy and not suffering from photosynthetic stress. Our study showed that 12 weeks of Mg deficiency significantly decreased the F_v/F_m from 0.82 to 0.64 and the F_v/F₀ from 4.08 to 2.98. This shows that Mg deficiency caused inactivation damage of PS II. A higher F₀ observed after six weeks of Mg deficiency is associated with the oxidative damage and loss of PS II reaction centers [54]. The energy-trapping efficiency in the PS II reaction centers was impaired, and antennae were partially disconnected from the centers [55].

The Φ_PSII is the operating efficiency of PS II and shows the proportion of light actually used for PS II photochemistry [56]. The decrease in F_v/F_m and Φ_PSII indicated that the photoactivation of PS II was inhibited by Mg deficiency resulting from the destruction of antennae pigments and the limitation of QA reoxidation by the decrease in or partial block of electron transport from PS II to PS I [57].

The decreased F_v/F₀, F_v/F_m, Φ_PSII, qP, and NPQ and increased non-light-induced non-photochemical fluorescence quenching (Y(NO)) contributed to the decreased Pn under Mg deficiency. Reduction in Pn and qP after Mg deficiency indicated that carbon assimilation was suppressed in banana leaves. Similar results were obtained in citrus [28], Sulla carnosa [58], and maize [13]. In addition, lower gs and higher C_i after Mg deficiency indicated that the reduction in CO₂ assimilation was primarily caused by non-stomatal factors [59]. Furthermore, the involvement of non-stomatal factors is also evident by the reduction in F_v/F_m, apparent photosynthetic quantum efficiency (AQY), and enzymatic activity as indicated by the carboxylation efficiency (CE). Similar previous studies showed a reduction in CO₂ assimilation in broad bean [60] and Pinus radiata [61] due to decreased gs under Mg deficiency.

The decline in Pn is also suggested as the photoprotection in banana leaves under Mg-deficiency conditions due to the dissipation of excess energy as heat through the xanthophyll cycle [62]. Banana leaves adapt to Mg deficiency by using only a fraction of the excitation energy for photochemical reactions. Amaxl, Amaxc, and LSP decreased while LCP and CCP increased in banana leaves, suggesting that Mg deficiency severely affected photosystem II. A possible reason is the inhibition of the NPQ pathway, effective in dissipating excess energy generated during electron transfer [63], as indicated by the reduction in Y(NPQ) and increase in Y(NO) (Figure 5). Decreased Amaxl and Amaxc indicated that the photosynthetic capacity was substantially reduced. The decreased LSP and increased LCP suggested that the range of light intensity suitable for carbon assimilation in banana leaves was also reduced. Moreover, the increased CSP and CCP showed that Mg deficiency narrowed the CO₂ concentration range suitable for carbon assimilation via photosynthesis. Thus, the overall reduction in carbon assimilation could be attributed to non-stomatal factors, restricted photosynthetic conditions, and metabolic shift to photoprotection. Previously, it was thought that a Mg deficit mainly affected electron transport to the dark processes, reducing CO₂ assimilation, but current research shows that Mg deficiency affects CO₂ assimilation prior to the light reactions [64].

Our results also indicated that foliar Mg application could partially recover the chlorophyll concentration and photosynthesis in leaves. Similar results have been reported in maize and alfalfa [13,65]. Furthermore, the photosynthesis rate, protection of the photosynthetic apparatus, and the photosynthesis capacity of the banana plants after foliar application of Mg were significantly higher than the Mg-deficient plants, indicating that foliar Mg application can partially recover overall photosynthesis. The recovery could be attributed to the high mobility and translocation of Mg to the Mg-deficient organs [66] and increased Mg concentration in all organs of the banana seedlings. The enhanced photosynthetic activity by foliar Mg is also due to higher Rubisco activity, as reported by Rodriguez et al. [67].

5. Conclusions

Mg deficiency for 6 and 12 weeks reduced the Mg concentration in banana seedlings and affected their growth and development. The reduction in Mg in leaves’ tissue caused pigment degradation and marginal chlorosis due to oxidative damage, whereas the root growth was more severely affected after Mg deficiency in banana seedlings. Foliar Mg application efficiently alleviated the physiological damage of photosystem II. This effect might be attributable to the increased Mg concentration in the tissue, pigment content restoration, and electron transfer recovery in leaves. Further research is necessary to understand if the conclusions here could be generalized to other plant species, and molecular analyses would further confirm the results to improve foliar fertilizer formulations and their practical application.