Gypsum application ameliorates morphological and photochemical damages provoked by Al toxicity in Vaccinium corymbosum L. cultivars

Abstract

BACKGROUND:

By acidity, Al³⁺ available form increases, being toxic for plants. Calcium amendments are widely used as an agronomic practice to reduce this effect.

OBJECTIVE:

To determine the gypsum application effect on leaf morphological and physiological features on three highbush blueberry cultivars growing in acid soil and toxic Al level.

METHODS:

Legacy and Brigitta, Al-resistant and Bluegold, Al-sensitive were grown in acid soil with 48% Al saturation and three gypsum concentrations (0.7, 1.4, and 2.8 g CaSO₄kg^–1) for 30 days. Chlorophyll a fluorescence measurements, photosynthesis, and photoprotective pigments were analyzed. Samples of leaves and roots were harvested, and Al, Ca, and S concentrations, antioxidant activity (RSA), lipid peroxidation (LP), and leaf anatomy were determined.

RESULTS:

Gypsum decreased leaves and roots Al concentration in all cultivars. Higher Ca leaves concentration in cultivars was observed compared to roots under gypsum treatment. Aluminum damages were observed in leaf thickness, improving anatomic features in cultivars by gypsum as well as a reduction of LP without changes in RSA. Chlorophyll levels changed differentially according to the cultivar and CaSO₄ dose, while antheraxanthin was higher in Legacy with gypsum. The Principal Component Analysis (PCA) scores plot showed that PC1 separated Brigitta from Legacy-Al resistant, while PC2 helped to discriminate Bluegold Al-sensitive cultivar with gypsum treatment from the rest of the samples, including Bluegold without gypsum.

CONCLUSIONS:

Gypsum amendment ameliorates leaf morphology alterations as well as photochemical and biochemical damages in highbush blueberry under Al-toxicity, being cultivar-dependent. The morphological parameters can be important features as Al-resistance anatomical markers in highbush blueberry.

Keywords

Acid soils aluminum calcium sulfate highbush blueberry physiological responses

1 Introduction

Acid soils represent around 70% potentially arable soils and 30–40% arable soil in the world, where aluminum (Al) toxicity is the main factor that limit crop production in these soils [1, 2]. In south-central Chile, most crop production is developed in acid soils, which derive from young volcanic ashes (Andisols). The main characteristics of these soils are low pH (from 4.5 to 5.5), high organic matter content, low phosphorus (P) and nitrogen (N) availability as well as low ion exchange, and high water-holding capacities [3, 4]. In these soils, acidification is favored by urea fertilization and heavy rains, especially in winter, which lixiviate the main cations from the soil exchange complex, replacing them by protons (H⁺) and acid cations, mainly Al and manganese (Mn) [5]. Under acidic conditions, Al^3 + available form increases, thus being toxic for plants [6]. The first effect of Al toxicity is root growth reduction, which can severely affect water and nutrient uptake, reducing crop yield [7, 8]. Root growth reduction is due to the Al interaction with the apoplastic side in the cell wall, plasma membrane, and cytoskeleton [9]. Although, it has been reported that Al-toxicity produces disturbances on the peripheral tissues, and roots thickening [10, 11], little information has been reported at shoot levels. Konarska [12] reported that Al exposure reduced the size and thickness of leaf blades due to a decrease in cell size, as well as an increase in the number of stomata from the abaxial epidermis, with a simultaneous reduction of their size. These effects caused a decrease in photosynthetic pigment levels and photosynthetic parameters. Similarly, a reduction in leaf area and chlorophyll content were found in cotton plants growing under Al-toxicity [13]. Likewise, several reports indicated that Al also affects the photosynthetic performance, reducing the stomatal conductance and electron transport rate [14 –16]. Moreover, Al induces reactive oxygen species (ROS) production, leading to oxidative stress in organelles and eventually provoking cell death [17, 18]. Studies in highbush blueberry have demonstrated that long-term exposure to Al-toxicity increased lipid peroxidation (LP) mainly in Al-sensitive cultivar, augmenting the radical scavenging activity (RSA) in response to Al-stress [11 , 15]. In other species such as pea (Pisum sativum), lipid peroxidation seems to be an Al-toxicity early symptom [19].

For overcoming Al phytotoxicity, calcium (Ca) amendments have been widely used as an agronomic practice to reduce this effect in acid soils [20 –22]. This strategy is commonly used by farmers for the production of different crop species, being lime and gypsum or phosphogypsum the most common Ca source [23 –25]. Among the Ca sources, gypsum has the advantage of ameliorating subsoil acidity with the surface application and thus representing a good nutrients source, such as Ca and S, as well as having higher solubility rates in soil solution [4 , 25–27]. Furthermore, it is reported that the strong complex formed between Al and sulfate (SO₄^2–) provokes both, decreased Al toxicity in soil solution and decreased mineral nutrients translocation towards the upper part of plants (stem and/or leaves) [4]. Despite a few reports about the effect of gypsum application on soil [27 –30], gypsum effects on plant metabolism and physiology are still poorly known.

Vaccinium corymbosum L. (highbush blueberry) is one commercially important berry crop with different cultivars sensitivity to toxic Al levels. Its fruit has exceptional flavor, nutritional properties, and antioxidant-richness; therefore, it is highly demanded as healthy food [31]. This crop is well adapted to soil acidity [32]; however, its sensitivity to the presence of Al-toxicity decreases its productivity substantially. Therefore, this study aimed to investigate gypsum amendment effects on morpho-anatomical features and physiological performance on three highbush blueberry cultivars growing in acid soil and toxic Al level.

2 Materials and methods

2.1 Plant material and experimental conditions

One-year-old highbush blueberry plants, from three contrasting cultivars in terms of Al resistance (Legacy and Brigitta as Al-resistant, and Bluegold as Al-sensitive) were used in this study. Plants with uniform size (30 cm high) were obtained from Berries San Luis, Lautaro (38°29 S and 72°23 W), La Araucania Region, Chile. The experiment was performed under controlled greenhouse conditions at 20–25°C, 16/8 h photoperiod (light/dark, respectively), 80% relative air humidity, and photosynthetic photon flux density (PPFD) of 300μmol photons m^–2 s^–1 as an average, according to Reyes-Díaz et al.[33]. Acid soil (Andisol) from the Gorbea serie [34] was used with pH (in water) 4.7 and Al saturation 48%. Previous to starting the experiment, the gypsum amendment was added to the soil sample, carefully mixed, and then incubated for four weeks, as described by Mora et al. [3]. Thereafter, plants were carefully washed with deionized water and transferred to pots (1 plant per pot) with 2 kg acid soil or incubated soil as follows: i) acid soil with a 48% Al saturation; ii) acid soil + Al + 0.7 g CaSO₄ kg^–1 of soil; iii) acid soil + Al + 1.4 g CaSO₄ kg^–1 of soil, and iv) acid soil + Al + 2.8 g CaSO₄ kg^–1 of soil (Table 1). Pots were daily irrigated to maintain soil field capacity during the experiment (30 days). Each treatment had ten replicates in a completely randomized design.

2.2 Soil chemical properties

The soil chemical properties were determined at the beginning and the end of the experiment (30 days), according to Sadzawka et al. [35] (Table 1). Soil pH was potentiometrically measured in a soil/water solution ratio (1:2.5). Sulfur (S) was extracted with Ca (H₂PO₄) [36] and analyzed by turbidometry [37]. Calcium was extracted with 1 M CH₃COONH₄ at pH 7.0 and analyzed by a simultaneous multi-element atomic absorption spectrophotometer (model UNICAM 969 Atomic absorption Spectrometer, England, UK). Exchangeable Al was extracted with 1 M KCl and analyzed by the same equipment.

2.3 Aluminum and calcium concentrations in leaves and roots

At 30 days of the experiment, mature expanded leaves of shoots (from the first to the fourth node) and roots were dried separately at 70°C in a forced air oven for 48 h. Then, leaves and roots were ashed at 500°C for 8 h and treated with 2M hydrochloric acid. Aluminum and Ca were quantified using a simultaneous multielement atomic absorption spectrophotometer (Model 969 Atomic Absorption Spectrometer, Unicam, Cambridge, UK) as described in Sadzawka et al. [38].

2.4 Sulfur concentration in leaves and roots

For S concentration analysis, plant tissues were collected as mentioned above. Dry leaves and roots were treated with 95% magnesium nitrate (MgNO₃×6H₂O) and ashed at 500°C for 8 h. Ashed samples were digested with 10 mL of 2M HCl for 60 min at 150°C before the addition of barium chloride (BaCl₂) and Tween-80. The resulting solution was measured in a UV/VIS spectrophotometer (UNICO^® 2800 UV/VIS, Spain) at 440 nm, as described by Sawdzaka et al. [38].

Table 1
Chemical properties of Gorbea soil series, at the end of the experiment (30 days). Values represent the average of five replicates±SE (n = 5). Different lower case letters indicate statistically significant differences (p≤0.05) among treatments within cultivars. Different upper case letters show differences (p≤0.05) among cultivars within treatments.

Legacy Brigitta Bluegold

Parameters Acid soil + Al at the beginning 0 0.7 1.4 2.8 0 0.7 1.4 2.8 0 0.7 1.4 2.8

doses (g CaSO₄ kg soil) + Al doses (g CaSO₄ kg soil) + Al doses (g CaSO₄ kg soil) + Al

pH (water) 4.7±0.12a 4.8±0.01aA 5±0.03aA 5±0.07aA 5±0.06aA 4.7±0aA 4.9±0.01aA 5±0.06aA 5±0.06aA 4.9±0.01aA 4.9±0.01aA 5±0.03aA 5±0.03aA

Al Sat (%) 48±3.95a 32±0.37bA 23±1.48cA 15±0.59dB 9±0.23eA 34±0.74bA 22±1.18cA 17±0.19dA 8±0.04eA 29±2.41bB 22±1.5cA 17±1.6dA 8±0.01eA

Ca (cmol+/kg) 0.3±0.03a 0.39±0.08aA 1±0.1bA 1.6±0.1cA 3.3±0.06dA 0.32±0.04dA 1.0±0.05cA 1.2±0.14bA 3.7±0.12aA 0.38±0.01dA 1.06±0.13cA 1.7±0.1bA 3.5±0.12aA

S (ppm) 34±1.45a 38±0.29bA 61±4.91cA 82±7dA 136±5eC 40±2.02bA 62±4.33cA 87±4.04dA 158±9.81eA 42±0.29bA 62±4.33cA 70±2.9dA 147±6eB

		Legacy	Brigitta	Bluegold
pH (water)	4.7±0.12a	4.8±0.01aA	5±0.03aA	5±0.07aA	5±0.06aA	4.7±0aA	4.9±0.01aA	5±0.06aA	5±0.06aA	4.9±0.01aA	4.9±0.01aA	5±0.03aA	5±0.03aA
Al Sat (%)	48±3.95a	32±0.37bA	23±1.48cA	15±0.59dB	9±0.23eA	34±0.74bA	22±1.18cA	17±0.19dA	8±0.04eA	29±2.41bB	22±1.5cA	17±1.6dA	8±0.01eA
Ca (cmol+/kg)	0.3±0.03a	0.39±0.08aA	1±0.1bA	1.6±0.1cA	3.3±0.06dA	0.32±0.04dA	1.0±0.05cA	1.2±0.14bA	3.7±0.12aA	0.38±0.01dA	1.06±0.13cA	1.7±0.1bA	3.5±0.12aA
S (ppm)	34±1.45a	38±0.29bA	61±4.91cA	82±7dA	136±5eC	40±2.02bA	62±4.33cA	87±4.04dA	158±9.81eA	42±0.29bA	62±4.33cA	70±2.9dA	147±6eB

2.5 Fluorescence of chlorophyll a parameters

Fluorescence of chlorophyll a parameters were determined in vivo using a portable pulse-amplitude modulated fluorometer (FMS 2; Hansatech Instruments, King’s Lynn, UK), to establish the PSII photochemical efficiency, according to Reyes-Díaz et al. [33]. The effective quantum yield (ΦPSII) and electron transport rate (ETR) were calculated as described by Maxwell and Johnson [39].

2.6 Photosynthetic and photoprotective pigments quantification

Photosynthetic and photoprotective pigments were extracted with 100% v/v acetone (HPLC grade) and determined, according to García-Plazaola and Becerril [40] by high-performance liquid chromatography (HPLC). The HPLC measurements were performed in an HPLC System Agilent technologies 1200 series, column C-18 Waters spherisorb 5.0μm ODS1 4.6×250 mm. Chlorophyll (Chl) a, b, α-carotene (α-Ca), violaxanthin (Vx), antheraxanthin (Ax), neoxanthin (Nx), and Lutein (Lt) were measured using pigment standards purchased from Sigma-Aldrich (Sigma Chemical Co. St. Louis, MO, USA).

2.7 Lipid peroxidation measurement

Lipid peroxidation (LP) was determined as an index of oxidative damage in plants and assessed in fresh samples (leaves and roots) by monitoring the thiobarbituric acid reacting substances (TBARS). In order to correct the interference produced by TBARS-sugar complexes, the absorbance was measured at 440, 532, and 600 nm according to a modified method [41]. The LP was expressed as nmol equivalents of malondialdehyde (MDA) concentration per gram of fresh weight (FW) in nmol MDA g^–1 FW.

2.8 Radical scavenging activity quantification

The radical scavenging activity (RSA) of roots and leaves was measured by free radical 1.1-diphenyl-2-picrylhydrazyl (DPPH) method, as described by Chinnici et al. [42]. The absorbance was measured at 515 nm using Trolox as standard. The RSA was expressed as microgram of Trolox equivalent per gram of fresh weight (μg TE g^–1 FW).

2.9 Morpho-anatomical analysis

Leaves were collected as described above and placed in a fixing solution; the central portion of leaves was fixed in formaldehyde, acetic acid, and ethanol (FAA) for 72 h and preserved in 70% ethanol (v/v). Transverse sections of 10μm were stained with safranine fast green and mounted in water-glycerol, then visualized by microscopy (Olympus CX31, Tokyo Japan) [43]. Finally, leaf morphology was evaluated using Image J software; the width of cell layers such as adaxial epidermis (ADE), abaxial epidermis (ABE), mesophyll (M), and palisade (P), and leaf thickness were measured.

2.10 Experimental design and statistical analyses

The experiment was completely randomized with 3 genotypes×4 treatments×5 replicates each. Pots with one plant each were changed every day to minimize positional effects. Measurements of chlorophyll a fluorescence parameters were performed at 0, 7, 15 and 30 days after gypsum application, whereas morpho-anatomical, chemical and biochemical quantifications were made at the end of the experiment (30 days). All data passed the normality and the equality of variance after the Kolmogorov-Smirnov test. Data were analyzed with a two-way analysis of variance (ANOVA) (where the factors were cultivars and treatments) for chemical and biochemical analyses and to a three-way ANOVA (where the factors were cultivars, treatments, and time) for photosynthetic parameters. Tukey test was applied to identify means with significant differences at the level of p≤0.05. Analyses were performed with Sigma Stat 2.0 software (SPSS, Chicago, IL).

The Pearson correlation analysis was carried out by t-test with a significance level of p≤0.05 to examine the relationships among variables. The Benjamini and Hochberg [44] false discovery rate control was used for correcting the resulting p-values, using R script displayed by the Rbio software (www.biometria.ufv.br). To reduce the dimensionality of data set, and identify the variables that explained a higher proportion of the total variance, which could provide insight into the effects of gypsum application on biochemical and physiological features, a multivariate analysis by Principal Components Analysis (PCA) was used in the Minitab^® 17 statistics program (Minitab Inc., Philadelphia).

3 Results

3.1 Soil chemical properties after gypsum treatment

Soil pH increased from 4.7 to 5.0 from the beginning until the end of the experiment in all amendment treatments (Table 1). Aluminum saturation in soil decreased, concomitant with gypsum amendment application in all cultivars, reaching a 6-fold decrease at the highest CaSO₄ dose (Table 1). Calcium concentration in soil increased 3-fold in the lowest gypsum dose, 5- and 12-fold in the highest doses in all cultivars (Table 1). Soil sulfur concentration augmented until 4.6-fold with the highest gypsum amendment dose in all cultivars (Table 1).

Fig.1

Aluminum, calcium and sulfur concentrations in leaves and roots of three cultivars of V. corymbosum subjected to acid soil with 48% aluminum saturation and different CaSO₄ treatments for 30 days. A and B: Al concentration in leaves and roots, respectively; C and D: Ca concentration in leaves and roots, respectively and E and F: S concentration in leaves and roots, respectively. Values represent means (n = 5)±SE. Different lower case letters indicate statistically significant differences (p≤0.05) among treatments within cultivars and plant tissues. Different upper case letters show differences (p≤0.05) among cultivars within treatments and plant tissues. Asterisk (*) indicates statistically significant differences between tissues (leaves and roots) for the same cultivar and treatments.

3.2 Aluminum, calcium, and sulfur concentrations in leaves and roots

A significant interaction was observed among organs, cultivars, and treatments for Al concentration (p≤0.001). The Al concentrations in leaves and roots of all cultivars showed a significant decrease (p≤0.05) when the amendment was applied (Fig. 1). In acid soil treatment, the roots of the three cultivars presented higher Al concentration than leaves (Fig. 1A, B). The leaves from Bluegold exhibited the lowest Al concentration than the other cultivars under acid soil treatment (Fig. 1A). When the highest dose of the amendment was applied, Al concentration in leaves decreased. Interestingly, in Brigitta leaves, a stronger reduction in Al levels (55%) was observed, followed by Legacy (27%) and Bluegold (23%) (Fig. 1A). In roots, the three cultivars diminished their Al concentration due to the effect of amendments when compared with non-calcium treated roots (acid soil + Al) (Fig. 1B). Legacy presented the highest Al reduction (around 86%) by the highest doses of amendments, whereas in Brigitta and Bluegold the decrease of Al was lower (62%) at the highest CaSO₄ application (Fig. 1B). Significant interactions for Ca concentration were observed between organs versus cultivars and cultivar versus treatments (p≤0.001). The Ca concentration in leaves of all cultivars was higher than in roots (p≤0.05), exhibiting increased levels of Ca when the amendment was applied (Fig. 1C, D). The Ca concentration in Bluegold and Legacy roots did not show any significant change when amendments were added. However, Brigitta roots gradually incremented Ca concentration with treatments (p≤0.05) regarding Al-treated plants (Fig. 1D).

A significant interaction among organs, cultivars, and treatments was observed in leaves and roots S concentration (p≤0.001). In general, S concentration in leaves was higher than in roots in all cultivars (Fig. 1E, F). Besides, S concentration was increased in leaves and roots of the three cultivars (p≤0.05) after the amendment application. An increase of S concentration (30%) was observed in Bluegold leaves with the lowest amendment dose concerning to acid soil without amendment (Fig. 1F). Among cultivars, Brigitta and Bluegold leaves presented higher S concentration than Legacy leaves (Fig. 1E).

3.3 Chlorophyll fluorescence parameters

A significant interaction between cultivars and time of measurements was observed for ΦPSII and ETR (p≤0.001). Legacy slightly decreased ΦPSII and ETR values under acid soil treatment, increasing these parameters with amendment application, being higher (32%) under 1.4 g CaSO₄ kg^–1 of soil treatment (Fig. 2). Brigitta increased ΦPSII and ETR with all amendment application through the time, whereas Bluegold decreased these parameters under acid soil treatment after seven days, reaching the initial values with CaSO₄ application (Fig. 2). NPQ did not vary among treatments and times in all cultivars (data not shown).

Fig.2

Changes in the effective quantum yield (ΦPSII) and electron transport rate (ETR) of three cultivars of V. corymbosum at different times (days) subjected to acid soil with 48% aluminum saturation and different CaSO₄ treatments for 30 days. Values represent means (n = 5)±SE. Asterisk (*) indicates statistically significant differences (p≤0.05) among treatments within cultivars.

3.4 Photosynthetic and photoprotective pigments

With respect to Chl a and Chl b, a significant interaction was observed between cultivar and treatments (p≤0.007 and p = 0.001, respectively). In Brigitta and Bluegold, total chlorophyll (Chl a+b), Chl a and Chl b concentrations increased with all amendment treatments, being around 43% at the highest CaSO₄ treatment respect to the acid soil alone (Fig. 3A-C). Bluegold showed a higher concentration of Chl a+b, Chl a, and b in all levels of gypsum application than Brigitta and Legacy (Fig. 3A-C). Chlorophyll a/b ratio did not change in any cultivar and treatments (Fig. 3D).

Fig.3

Chlorophyll (Chl) a and b in V. corymbosum L. grown in acid soil with different CaSO₄ amendment at 30 days. Acid soil (48% aluminum saturation). A: Chlorophyll a + b; B: Chlorophyll a; C: Chlorophyll b and D: Chlorophyll a/b. Values represent means (n = 5)±SE. Different lower case letters indicate statistically significant differences (p≤0.05) among treatments within cultivars. Different upper case letters show differences (p≤0.05) among cultivars within treatments.

Fig.4

Photoprotective pigments of highbush blueberry cultivars grown in an acid soil (48% aluminum saturation) under different CaSO₄ treatments at 30 days. Values represent means (n = 5)±SE. A: Total photoprotective pigments; B: α-carotene; C: Lutein; D: Neoxanthin; E: Violaxanthin; F: Anteraxanthin; G: Ant/Viol. Different lower case letters indicate statistically significant differences (p≤0.05) among treatments within cultivars. Different upper case letters show differences (p≤0.05) among cultivars within treatments.

Carotenoids pool was significantly increased in Bluegold (around 2-fold) under gypsum applications compared to the acid soil (Fig. 4A). This increment was lower in Brigitta, while Legacy levels remained unchanged (Fig. 4A). The α-carotene significantly enhanced (2.6-fold) in Bluegold by gypsum application in comparison to acid soil, staying constant in Brigitta and Legacy (Fig. 4B). Lutein concentration showed a significant increase (p≤0.05) when the amendment was applied in Brigitta and Bluegold cultivars (Fig. 4C). In the treatment without amendment, the highest Lutein concentration was observed in Legacy, followed by Bluegold, and the lowest in Brigitta (Fig. 4C). The xanthophyll cycle (neoxanthin, violaxanthin, and antheraxanthin) was enhanced in Brigitta and Bluegold after the amendment application, whereas in Legacy, these compounds did not change (Fig. 4D-F). The ratio of antheraxanthin and violaxanthin showed the highest value in Brigitta under acid soil, decreasing with amendment treatments. A similar tendency was observed in Legacy, whereas in Bluegold, this ratio augmented concomitant to the amendment respect to acid soil (Fig. 4G).

3.5 Lipid peroxidation and radical scavenging activity

There is significant interaction among cultivars, treatments, and organs of V. corymbosum (p≤0.001). At the end of the experiment, leaves LP values were higher in plants without CaSO₄ and with the lowest CaSO₄ dose, whereas the highest LP value was found in Legacy (Fig. 5A). In roots, Bluegold showed the highest LP in the treatment without amendment (Fig. 5B).

Fig.5

Lipid peroxidation measured as malondialdehyde (MDA) concentration in highbush blueberry cultivar leaves (A) and roots (B), grown for 30 days under different treatments in an acid soil (48% aluminum saturation). Values are means±SE (n = 5). Different lower case letters indicate statistically significant differences (p≤0.05) among treatments within cultivars. Different upper case letters show differences (p≤0.05) among cultivars within treatments.

Regarding the radical scavenging activity (RSA), a significant interaction was observed among cultivars, treatments, and organs (p = 0.011). Generally, higher leaves RSA was observed in Bluegold (Fig. 6A). The highest RSA was verified in Legacy roots, followed by Brigitta and Bluegold (Fig. 6B). The RSA was higher, around 2-fold increase, in Bluegold and Brigitta leaves than in roots, whereas in Legacy, this increase was only 1.3-fold (Fig. 6).

3.6 Morpho-anatomical analysis

Brigitta leaves thickness, and palisade mesophyll cells were higher than Bluegold and Legacy without amendments (p≤0.005), whereas an increase in all cultivars was found after gypsum application (Table 2 and Fig. 7). In Brigitta and Bluegold, a significant increase was exhibited (Table 2 and Fig. 7) in the upper epidermis cells (about 26%) with amendments (around 28%) with respect to non-amendment plants. It was also observed that Brigitta palisade mesophyll cells increased by 28% with 2.8 g kg^–1 CaSO₄ and 49% in Legacy with 1.4 g kg^–1 CaSO₄, respect to their values in acid soil. Spongy parenchyma thickness increased with increasing dose of CaSO₄, as follows: 45% in Legacy and Brigitta, and 24% in Bluegold (Table 2 and Fig. 7).

Fig.6

Leaf and root-scavenging capacity, measured as Trolox equivalents (TE), of highbush blueberry cultivars grown in an acid soil (48% aluminum saturation), under different CaSO₄ treatments at 30 days. (A): leaves and (B): roots. Values are an average of five replicates±SE. Different lower case letters indicate statistically significant differences (Tukey’s HSD at P≤0.05) among treatments for the same cultivar. Different upper case letters indicate differences (Tukey’s HSD at P≤0.05) among cultivar and similar treatment. Asterisk (*) indicates statistically significant differences between tissues (leaves and roots) for the same cultivar and treatments.

3.7 Correlation analysis of variables

To reduce the dimensionality of the data set and to identify significant associations, the Pearson correlation was used (Fig. 8). For the Legacy cultivar, some correlations were expected like the positive correlations between palisade parenchyma thickness (r = 0.97) and spongy parenchyma thickness (r = 0.97) with leaf thickness (Fig. 8A). Others were less intuitive, such as NPQ strongly and positively correlated with sulfur levels in leaves at all times (r > 0.96). It is worth mentioning that Ca levels in leaf and root were weakly correlated with other traits (Fig. 8A).

Table 2
Morphometric leaf parameters of three V. corymbosum cultivars under an acid soil with 48% aluminum saturation and different CaSO₄ treatments at the end of the experiment (30 days)

Treatments Cultivars

Legacy Brigitta Bluegold

Leaf thickness (μm)

Acid soil + Al 10.43±0.66 Bd 14.63±0.01Ac 11.1±0.13Bc

Acid soil + Al+ 0.7 g Ca SO₄kg^–1 11.94±0.70 Bc 18.16±0.29 Ab 11.21±0.1 Bc

Acid soil + Al+ 1.4 g Ca SO₄ kg^–1 17.33±0.14 Ba 20.21±0.23 Aa 12.86±0.13 Cb

Acid soil + Al + 2.8 g Ca SO₄ kg^–1 15.75±0.06 Bb 20.98±0.93 Aa 15.31±0.21 Ba

Upper epidermis thickness (μm)

Acid soil + Al 1.23±0.01 Aa 1.27±0.06 Ac 1.30±0.01 Ac

Acid soil + Al + 0.7 g Ca SO₄kg^–1 1.31±0.04 Aa 1.44±0.04 Abc 1.40±0.04 Abc

Acid soil + Al + 1.4 g Ca SO₄ kg^–1 1.30±0.06 Ba 1.48±0.06 ABb 1.55±0.08 Aab

Acid soil + Al + 2.8 g Ca SO₄ kg^–1 1.25±0.09 Ba 1.77±0.07 Aa 1.72±0.06 Aa

Lower epidermis thickness (μm)

Acid soil + Al 0.91±0.07 Aa 0.96±0.03 Ac 0.97±0.01 Aa

Acid soil + Al + 0.7 g Ca SO₄kg^–1 1.12±0.02 Aa 1.04±0.04 Abc 1.06±0.01 Aa

Acid soil + Al + 1.4 g Ca SO₄ kg^–1 1.09±0.05 Aa 1.27±0.06 Aab 1.01±0.03 Aa

Acid soil + Al + 2.8 g Ca SO₄ kg^–1 1.16±0.06 Ba 1.49±0.17 Aa 0.91±0.02 Ca

Palisade parenchyma thickness (μm)

Acid soil + Al 2.42±0.10 Cc 4.96±0.09 Ac 3.37±0.09 Ba

Acid soil + Al + 0.7 g Ca SO₄kg^–1 3.26±0.15 Bb 5.57±0.00 Ab 3.37±0.42 Ba

Acid soil + Al + 1.4 g Ca SO₄ kg^–1 4.79±0.04 Ba 6.64±0.08 Aa 3.68±0.05 Ca

Acid soil + Al + 2.8 g Ca SO₄ kg^–1 3.70±0.20 Bb 6.87±0.17 Aa 3.40±0.12 Ba

Spongy parenchyma thickness (μm)

Acid soil + Al 4.51±0.18 Bb 6.06±0.26 Ad 5.44±0.10 ABc

Acid soil + Al + 0.7 g Ca SO₄kg^–1 4.73±0.23 Cb 7.03±0.33 Ac 5.82±0.28 Bc

Acid soil + Al + 1.4 g Ca SO₄ kg^–1 9.09±0.34 Aa 9.15±0.28 Ab 6.45±0.20 Bb

Acid soil + Al + 2.8 g Ca SO₄ kg^–1 8.19±0.24 Ca 11.01±0.29 Aa 7.16±0.49 Ba

Treatments	Cultivars
Leaf thickness (μm)
Acid soil + Al	10.43±0.66 Bd	14.63±0.01Ac	11.1±0.13Bc
Acid soil + Al+ 0.7 g Ca SO₄kg^–1	11.94±0.70 Bc	18.16±0.29 Ab	11.21±0.1 Bc
Acid soil + Al+ 1.4 g Ca SO₄ kg^–1	17.33±0.14 Ba	20.21±0.23 Aa	12.86±0.13 Cb
Acid soil + Al + 2.8 g Ca SO₄ kg^–1	15.75±0.06 Bb	20.98±0.93 Aa	15.31±0.21 Ba
Upper epidermis thickness (μm)
Acid soil + Al	1.23±0.01 Aa	1.27±0.06 Ac	1.30±0.01 Ac
Acid soil + Al + 0.7 g Ca SO₄kg^–1	1.31±0.04 Aa	1.44±0.04 Abc	1.40±0.04 Abc
Acid soil + Al + 1.4 g Ca SO₄ kg^–1	1.30±0.06 Ba	1.48±0.06 ABb	1.55±0.08 Aab
Acid soil + Al + 2.8 g Ca SO₄ kg^–1	1.25±0.09 Ba	1.77±0.07 Aa	1.72±0.06 Aa
Lower epidermis thickness (μm)
Acid soil + Al	0.91±0.07 Aa	0.96±0.03 Ac	0.97±0.01 Aa
Acid soil + Al + 0.7 g Ca SO₄kg^–1	1.12±0.02 Aa	1.04±0.04 Abc	1.06±0.01 Aa
Acid soil + Al + 1.4 g Ca SO₄ kg^–1	1.09±0.05 Aa	1.27±0.06 Aab	1.01±0.03 Aa
Acid soil + Al + 2.8 g Ca SO₄ kg^–1	1.16±0.06 Ba	1.49±0.17 Aa	0.91±0.02 Ca
Palisade parenchyma thickness (μm)
Acid soil + Al	2.42±0.10 Cc	4.96±0.09 Ac	3.37±0.09 Ba
Acid soil + Al + 0.7 g Ca SO₄kg^–1	3.26±0.15 Bb	5.57±0.00 Ab	3.37±0.42 Ba
Acid soil + Al + 1.4 g Ca SO₄ kg^–1	4.79±0.04 Ba	6.64±0.08 Aa	3.68±0.05 Ca
Acid soil + Al + 2.8 g Ca SO₄ kg^–1	3.70±0.20 Bb	6.87±0.17 Aa	3.40±0.12 Ba
Spongy parenchyma thickness (μm)
Acid soil + Al	4.51±0.18 Bb	6.06±0.26 Ad	5.44±0.10 ABc
Acid soil + Al + 0.7 g Ca SO₄kg^–1	4.73±0.23 Cb	7.03±0.33 Ac	5.82±0.28 Bc
Acid soil + Al + 1.4 g Ca SO₄ kg^–1	9.09±0.34 Aa	9.15±0.28 Ab	6.45±0.20 Bb
Acid soil + Al + 2.8 g Ca SO₄ kg^–1	8.19±0.24 Ca	11.01±0.29 Aa	7.16±0.49 Ba

Values represent the average of five replicates±SE (n = 5). Different lower case letters indicate statistically significant differences (p≤0.05) among treatments within cultivars and plant tissue. Different upper case letters show differences (p≤0.05) among cultivars within treatments and plant tissues.

The highest number of significant correlations were found for Brigitta cultivar (70 correlations with a p-value below 0.05) (Fig. 8B). The correlation analysis showed that in Brigitta, Al levels in leaf were strongly and negatively correlated with two important pigments involved with the protection of the photosynthetic apparatus, which are α-carotene (r = –0.99) and violaxanthin (r = –0.99) (Fig. 8B). In Brigitta, the highest correlation was exhibited between epidermis thickness parameters and foliar Ca levels (r > 0.95) (Fig. 8B). When we analyzed the Bluegold cultivar, similarly to what was found in Brigitta, photosynthetic pigments such as Chl a, Chl b and α-carotene have a strong and positive correlation (r > 0.95) with most of the xanthophyll cycle pigments analyzed in this study, such as neoxanthin, violaxanthin, and anteraxanthin. On the other hand, only in Bluegold, the foliar lipid peroxidation was negatively correlated with these pigments. Besides, only in the Brigitta cultivar, a remarkably positive correlation was observed between Ca and S foliar levels with root sulfur levels (r = 0.99).

Fig.7

Morpho-anatomical features of three highbush blueberry cultivars exposed under acid soil (48% aluminum saturation), and different CaSO₄ treatments for 30 days. The image of the transverse section of the leaves shows leaf thickness (LT), upper epidermis thickness (UET), lower epidermis thickness (LET), Palisade parenchyma thickness (PPT), and Spongy parenchyma thickness (SPT). Scale bars represent 5μm.

Fig.8

Correlation matrices based on Pearson's correlation coefficients between physiological and biochemical features of leaves and roots as well as leaves morphological traits of blueberry cultivars with contrasting Al resistance (Legacy A, Brigitta B, and Bluegold C), growing under increasing CaSO₄ treatments (acid soil + Al, 0.7, 1.4 and 2.8 g CaSO₄ kg^–1 of soil + Al). Physiological parameters analyses were performed at acid soil and Ca-treated plants on different days (0, 7, 15, 30 days). Significant correlation coefficients (p_adj ≤0.05) are indicated in bold. Each square represents three biological replicates average, with positive and negative correlations being distinguished by blue and red, respectively. Abbreviations: electron transport rate (ETR); non-photochemical quenching (NPQ); aluminum (Al); calcium (Ca); sulfur (S); radical scavenging activity (RSA); chlorophyll (Chl); upper epidermis thickness (UET); lower epidermis thickness (LET); palisade parenchyma thickness (PPT) and spongy parenchyma thickness (SPT).

3.8 Principal component analysis (PCA) of the physiological and biochemical measurements from leaves and roots

In order to investigate the possible common factors that would explain the observed correlations, PCA analysis was performed based on all measured traits from leaves and roots of the three blueberry cultivars (Legacy, Bluegold and Brigitta) (Fig. 9). For this comparison, data obtained for all traits were averaged and normalized. The first two dimensions resumed a higher part of the total variance (66.9%). The first principal component (PC1) and the second principal component (PC2) accounted for 37.7% and 29.2% total variation, respectively (Fig. 9A). The PCA scores plot showed that PC1 separated Brigitta from Legacy Al-resistant, while PC2 helped to discriminate Bluegold Al-sensitive cultivar with gypsum treatment from the rest of the samples, including Bluegold without gypsum (Fig. 9A). Overall, according to the percentage of total variability explained by PC1, the global changes between these two cultivars may be related to changes in the physiological (ETR and NPQ) and epidermis thickness parameters showed by Brigitta in contrast to biochemical changes (neoxanthin, violaxanthin and antheraxanthin) displayed by Legacy (Fig. 9B).

Fig.9

Principal component analysis (PCA) representing physiological and biochemical features from leaves and roots as well as leaves morphological traits of three blueberry cultivars with contrasting Ca tolerance (Legacy, Bluegold, and Brigitta), growing under increasing CaSO₄ treatments (acid soil + Al, 0.7, 1.4 and 2.8 g CaSO₄ kg^–1 of soil + Al). The analysis of physiological parameters was performed in plants grown in acid soil at 48% aluminum saturation. (A) A score plot of the first component (PC1) against the second component (PC2); it shows the averages of the whole data pool analyzed in the investigated samples. (B) The loadings plot obtained from the resulting distribution of physiological and biochemical data. Numbers in parentheses give the variation percentage explained by the first and the second principal component, respectively. Circle and text colors indicate the cluster assigned using hierarchical clustering. Abbreviations: electron transport rate (ETR); non-photochemical quenching (NPQ); aluminum (Al); calcium (Ca); sulfur (S); radical scavenging activity (RSA); chlorophyll (Chl); upper epidermis thickness (UET); lower epidermis thickness (LET); palisade parenchyma thickness (PPT) and spongy parenchyma thickness (SPT).

On the other hand, the PC2 clearly separated cultivar Bluegold from all other cultivars separated along PC1. Furthermore, it revealed an interesting ungrouping between acid soil and treatments growing under increasing CaSO₄ doses (0.7, 1.4, and 2.8 g CaSO₄ kg^–1 of soil) (Fig. 9A). Thus, this result suggests a great degree of Bluegold sensitivity when exposed to increasing doses of CaSO₄. It should also be highlighted that results obtained in the PC2 analysis also revealed that pigments such as Chl a, Chl b and carotene displayed a major percentage of the total variability (Fig. 9B). This indicates that the effect of the increasing Ca doses on the photosynthetic pigments was important for Bluegold plants.

4 Discussion

4.1 The CaSO₄ effect on interactions between other elements

It is widely known that Al-toxicity in acid soils can be decreased by calcareous amendments applications, such as gypsum (CaSO₄) [3 , 45–47]. Our results confirm this evidence in the three blueberry cultivars subjected to gypsum amendments, where Al soil saturation, as well as Al-concentrations of leaves and roots, decreased concomitantly with the increase of the amendment application (Table 1, Fig. 1). Studies performed in barley (Hordeum vulgare) under Al-toxicity showed that Ca addition reduced Al-toxicity due to Al concentration and lipid peroxidation reduction, which increased antioxidant enzyme activity as well as Ca concentration compared with the Al-treatment alone. Thus, suggesting that Ca supplementation could be related to less Al-uptake in barley plants [48]. This CaSO₄ application effectiveness in amelioration of Al-toxicity was also verified in lettuce under field conditions [30].

Furthermore, except for Al-accumulator plants, it has been reported that Al concentrates more in roots than in leaves [12]. In blueberry, Al-accumulation in different organs depended on the cultivar and the treatments. The Al-sensitive cultivar Bluegold accumulated more Al in roots than in leaves with the exception of the highest amendment supply. This high Al-root concentration in this cultivar provoked high oxidative stress as indicated by the higher LP (Figs. 1 and 5). Similar results have been found in the nutrient solution experiment, where Bluegold accumulated higher Al concentration in roots than in shoots when gypsum amendments were applied [15]. In addition, negative correlations between Al and Ca concentrations were found in Brigitta and Bluegold leaves and roots (r = around – 0.6 and – 0.84, p≤0.05, respectively), whereas Legacy leaves showed a lower correlation (r =– 0.46; p≤0.05) and in roots, no statistically significant correlation was found. These results indicated that in Brigitta and Bluegold, but not in Legacy, a decrease of Al^3 + interacting with Ca^2 + improved Ca uptake from the gypsum amendments (Fig. 1). High external Ca, in cytosolic root cells, reduces Al^3 + and favors the Ca uptake. Similar behavior has been proposed for K in Arabidopsis thaliana [49].

Based on tissues Ca concentrations, blueberry can be considered as a calcifuge species [29]. They reported that healthy blueberry plants leaves have Ca levels that vary from 3.0 to 8.0 g kg^–1 DW. Our values are in the range reported, depending on cultivar, being Ca levels higher in Bluegold leaves compared to other cultivars (Fig. 1). In rice (Oryza sativa), the application of amendments increased soil pH and reduced Al toxicity, improving growth and development due to the addition of Ca and other nutrients such as S [22]. Similar results have been reported in other crop species [26]. In the current study, blueberry cultivars exhibited negative correlation (p≤0.05) between Al and S concentration in Brigitta roots and leaves (r = – 0.82 and – 0.90, respectively) and Bluegold (r = – 0.79 and – 0.50, respectively), whereas this correlation was present (r = – 0.47) only in Legacy leaves. Contrarily, Mora et al. [7] reported that a relationship between S and Al contents in roots was positively correlated (r = 0.683) in ryegrass (Lolium multiflorum) plants under Taylor and Foy nutrient solution. In our work, it is remarkable that Bluegold and Brigitta showed a higher S concentration in leaves as compared to roots. Reyes-Díaz et al. [15] observed a high S concentration in Bluegold leaves subjected to Hoagland nutrient solution with toxic Al, under acidic conditions. In our study, a positive effect of CaSO₄ application was observed, increasing Ca and S contents in blueberry leaves and roots. Furthermore, studies performed in wheat and ryegrass indicates that Ca content in leaves increased dramatically according to CaSO₄ doses increase [3, 28]. Amendments can increase Ca and SO₄ content in the soil, making it available to plants and reducing the Al toxic-forming Ca-Al complex. Unexpectedly, although Bluegold is an Al-sensitive cultivar, it showed a higher Ca concentration in leaves than in roots, differently than in Legacy and Brigitta, which are more Al-resistant.

4.2 Photochemical responses of PSII to CaSO₄ and Al levels

The maximum photochemical efficiency of PSII (Fv/Fm) was not affected by any of the Al and CaSO₄ treatments (data not shown). Similar results were obtained in blueberry subjected to similar treatments in hydroponic Hoagland nutrient solution [15]. In this study, gypsum application enhanced the ΦPSII in the Al-resistant cultivars (Brigitta and Legacy) compared to the values of toxic Al in acid soil without amendments, whereas in Al-sensitive cultivars (Bluegold), ΦPSII values remained constant or decreased at the end of treatment with toxic Al alone (Fig. 2). This result confirms that this cultivar is more sensitive under Al-stress than the other cultivars, as reported previously [14, 33]. In Glycine max varieties, fluorescence parameters (Fv/Fm and ΦPSII) were reduced under toxic Al [50]. To counteract the damage caused by stress factors such as excessive light as well as Al-toxicity, plant tissues possess different photoprotective mechanisms, which include the xanthophyll cycle (XC) [51, 52]. These authors reported that in Citrus spp. exposed to Al-toxicity conditions antheraxanthin (A) and zeaxanthin (Z) increased in Al-treated leaves. In Al-resistant Legacy cultivar, a slight conversion of violaxanthin into antheraxanthin by gypsum supply was found, as compared with other cultivars (Fig. 4E and F). In addition, gypsum application increased all photoprotective pigments compared to Al-treatment. Chen et al. [51] reported that under high light and Al-toxicity, the increasing conversion of V to A and Z might help to quench ¹O₂ augmented in Al-treated leaves due to enhanced closure of PSII in Citrus. Nonetheless, in our experiment, we observed a higher increment in lutein pigment by gypsum application, especially in Bluegold compared to the acid soil and other cultivars (Fig. 4). This may be associated with a high PSII sensitivity to the Al-stress of Bluegold, due to the fact that lutein participates in the structural stabilization of light-harvesting antenna proteins, quenching ³Chl states. Besides, lutein has been proposed to be involved in the quenching of ¹Chl [53].

A typical symptom of oxidative stress is the increased lipid peroxidation (LP), which is considered a general index of oxidative membrane injury [19, 54]. Another study evidenced that LP is also a manifestation of Al toxicity in highbush blueberry [14]. In our research, the CaSO₄ application caused a LP decrease in roots and leaves of the three different cultivars with the exception of Legacy roots (Fig. 5). In Bluegold, this decrease was related to a significant increase of Lutein content and RSA (Figs. 4 and 6). Also, Maxwell and Johnson [39] indicate that high RSA can help minimize thylakoid damages, increasing ETR, which induces an increment of photochemical performance such as shown in our study, where Bluegold increased RSA, maintaining ETR compared to Brigitta and Legacy with Ca supply (Figs. 2 and 6). Gypsum application on Brigitta Al-resistant cultivar enhanced ΦPSII and ETR under Al treatments, unlike Bluegold Al-sensitive, where this parameter remained constant, decreasing only by Al alone.

4.3 Gypsum effects on morphological traits in blueberry leaves under Al toxicity

Aluminum toxic levels in soil promoted alterations in leaves morphological features. In our study, we observed a negative correlation between Al levels in soil and leaf thickness in the Al-tolerant cultivars (Brigitta and Legacy). The highest leaf thickness in Brigitta coincided with an augment of the palisade and spongy parenchyma thickness (Table 2 and Fig. 7). Our results agree with those reported by Konarska [12] with a reduction of morphological characteristics. In addition, studies performed in Eucalyptus revealed changes in leaf morphology under Al toxicity, such as thickness reduction of leaf epidermis and palisade layers [55]. Calcium concentration in Al-resistant Brigitta cultivar leaves generally correlated positively and significantly with leaf thickness, in each cellular layer evaluated (Table 2 and Fig. 7). These correlations could be an important trait for Al-resistance observed in Brigitta, and may be used as Al resistance anatomical markers for sensitivity in highbush blueberry cultivars.

4.4 Principal component analyses of metabolic, morphologic and physiological traits

Into a broader metabolic, morphological, and physiological context, PCA analysis suggests that Al-sensitive and resistant cultivars display distinct response mechanisms (Fig. 9A). The Al responses presented by the Brigitta cultivar were related to changes in Chlorophyll a fluorescence and morphological parameters, such as parenchyma thickness (Fig. 9B). It has been shown that Al toxic levels induce anatomical changes in plant tissues, reducing cell elongation, and division [56]. In sunflower plants, Al stress increased leaf blade thickness and parenchyma layers [57]. Thus, anatomical changes observed in Brigitta can be important traits of tolerant plants and may be used as anatomical markers of Al tolerance for sensible cultivars such as Bluegold.

On the other hand, in the Legacy cultivar, this tolerance was more associated with a higher Al accumulation in leaves and roots, followed by a clear LP increase for both organs (Fig. 9B). It is well known that plasma membrane is one of the primary Al-stress targets at the cellular level [58] and Al important effects appear on plasma membrane structure and function. Accordingly, high lipid peroxidation was found in all CaSO₄ treatments of Legacy cultivar. This effect may be because Legacy displayed more elevated Al accumulation in leaves than the other cultivars, even after the application of CaSO₄. As regards the Al-sensitive Bluegold cultivar, PCA revealed a clear separation between Al and calcium treatments (Fig. 9B). These results suggested that Bluegold cultivar has a positive response to CaSO₄ applications. Thus, compared to other cultivars, Bluegold might have a differential Al uptake by roots, which are revealed by a strong antagonistic group between Al levels in leaves and roots with calcium treatments (Fig. 9B). Genotypic differences have been characterized in Al uptake and accumulation in other crops like maize [59], wheat [60], and pigeon pea [61].

In conclusion, the gypsum amendment ameliorates leaf morphology alterations as well as photochemical and biochemical damages in highbush blueberry under Al-toxicity. In general, a compensatory effect was observed after the amendment application, increasing all evaluated parameters, particularly in the Brigitta cultivar with 1.4 g CaSO₄ kg^–1 supply. Gypsum application reverses the strong and negative correlation between Al content in leaves and leaf thickness, recovering the morphology and decreasing mesophyll compaction in leaves of all cultivars. Thus, morphological parameters can be important traits as anatomical markers of Al-resistance for Al-sensitive cultivars such as Bluegold.

Conflict of interest

The authors have no conflict of interest to report.

Funding

The authors report no funding.

Footnotes

Acknowledgments

We would like to thank PAI 80160036 and FONDECYT 11080231 Project for the financial support of this work and Dr. Helen Lowry for revising the language to the manuscript. E. Alarcon was supported by a Ph.D. fellowship MECESUP-UFRO 0601 and UFRO scholarship. Research fellowships granted by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to ANN and Fundação de Amparo á Pesquisa do Estado de Minas Gerais (FAPEMIG) to FMOS are also gratefully acknowledged.

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Gypsum application ameliorates morphological and photochemical damages provoked by Al toxicity in Vaccinium corymbosum L. cultivars

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1 Introduction

2 Materials and methods

2.1 Plant material and experimental conditions

2.2 Soil chemical properties

2.3 Aluminum and calcium concentrations in leaves and roots

2.4 Sulfur concentration in leaves and roots

2.6 Photosynthetic and photoprotective pigments quantification

2.7 Lipid peroxidation measurement

2.8 Radical scavenging activity quantification

2.9 Morpho-anatomical analysis

2.10 Experimental design and statistical analyses

3 Results

3.1 Soil chemical properties after gypsum treatment

3.3 Chlorophyll fluorescence parameters

4.1 The CaSO4 effect on interactions between other elements

4.2 Photochemical responses of PSII to CaSO4 and Al levels

4.3 Gypsum effects on morphological traits in blueberry leaves under Al toxicity

4.4 Principal component analyses of metabolic, morphologic and physiological traits

Conflict of interest

Funding

Footnotes

Acknowledgments

References

4.1 The CaSO₄ effect on interactions between other elements

4.2 Photochemical responses of PSII to CaSO₄ and Al levels