Light intensity and sulfur deficiency modulate growth and water dynamics in broccoli plants via aquaporin regulation

Abstract

• Sulfur plays a critical role in plant secondary metabolism, particularly in the biosynthesis of glucosinolates, where it functions as a core structural element and participates in molecular regulatory mechanisms. Moreover, sulfur metabolism is intricately connected to nitrogen assimilation, highlighting its multifaceted role in plant physiological processes. Light, another key abiotic determinant, directly modulates crop productivity, with light intensity governing essential processes such as growth kinetics and photosynthetic efficiency.

• This study aims to elucidate the effects of light stress and sulfur deficiency on broccoli (Brassica oleracea var. italica) growth and water dynamics under controlled environment conditions, both individually and in combination, to identify the physiological and molecular mechanisms activated in response to these stressors.

• The results revealed that sulfur deficiency has a stronger impact on plant water relations than light stress, while light stress mainly affects photosynthetic activity and biomass accumulation. Combined stresses lead to more pronounced physiological responses, including distinct aquaporin regulation patterns that differ from single stress treatments.

• These findings suggest a compensatory mechanism that helps maintain water balance, highlighting the complex interplay between sulfur availability, light intensity, and plant adaptation strategies.

Combined light stress and sulfur deficiency intensify broccoli adaptive responses, triggering distinct aquaporin expression patterns and highlighting a key compensatory mechanism for maintaining water homeostasis.

INTRODUCTION

Abiotic stresses can drastically reduce crop production, both qualitatively and quantitatively (Kopecká et al. ²⁰²³), and is an increasing problem due to climate change (Pachauri & Reisinger ²⁰⁰⁷). Abiotic stress affects both spring and winter crops, including Brassica oleracea L. var. italica (broccoli), an important species in European agriculture. Broccoli is a key source of affordable nutrients, containing minerals and bioactive compounds, such as phenolic acids and glucosinolates (Nagraj et al. ²⁰²⁰). Glucosinolates are primarily found within the Brassicaceae, although they are not exclusive to this family (Kjær 1974). These bioactive compounds have fungicidal, bactericidal, bioherbicidal, antioxidant, and antiproliferative activity, making them essential secondary metabolites with important defence action in plants (Vig et al. ²⁰⁰⁹). The concentrations and profile of glucosinolates vary across species, cultivar, and plant tissue (Fahey et al. ²⁰⁰¹), glucobrassicin and glucoraphanin being some of the major glucosinolates present in broccoli (Rangkadilok et al. ²⁰⁰²; Horbowicz ²⁰⁰³; Blažević et al. ²⁰²⁰). They share a basic structure, with a thioglucose moiety, a sulfonated oxime, and a side chain derived from amino acids (Halkier & Du ¹⁹⁹⁷). Depending on the precursor amino acid, glucosinolates can be classified into aliphatic, indolic, or benzenic (Chhajed et al. ²⁰²⁰).

Until a few decades ago, the quantities of sulfur necessary to satisfy crop needs were easily achieved, sulfur being regularly reintegrated into soils together with phosphoric and potassium fertilization. Industrial pollution from coal combustion also contributed to plant sulfur through aerial deposition. Recently, a widespread reduction in sulfur has begun to occur in soils. The global decline in atmospheric sulfur deposition driven by stricter emission regulations, along with reduced application of sulfur‐based fungicides, the replacement of sulfur‐containing fertilizers with new‐generation alternatives, reduced sulfur recycling from livestock manure, widespread cultivation of high‐yielding/nutrient‐demanding crop varieties, and the intensification of agricultural practices have collectively contributed to widespread sulfur deficiency in agroecosystems. These factors have disrupted the sulfur balance in many soils, a problem further exacerbated when crop residues are removed together with harvested products, limiting sulfur replenishment of the soil (Jamal et al. ²⁰¹⁰).

Sulfur plays a crucial role in glucosinolate biosynthesis, not only as a key component of their core structure but also as an integral part in molecular regulation of the biosynthetic process (Falk et al. ²⁰⁰⁷). Sulfur, primarily taken up as sulfate (SO₄ ²⁻), is absorbed through SULTR transporters located in epidermal and cortical membranes (Takahashi et al. ²⁰¹¹). These transporters are regulated by sulfate availability. Once absorbed, sulfate (which is highly mobile) is loaded into the xylem or stored in vacuoles (Maathuis & Diatloff ²⁰¹³). It can be either directly incorporated in a reaction termed ‘sulfation’ or used as a substrate for synthesis of cysteine and methionine after a multistep reduction to sulfide (Lewandowska & Sirko ²⁰⁰⁸). These two amino acids present chemical groups that are extremely important in the formation of protein structures, modulation of protein activity, and glucosinolate biosynthesis (Fahey et al. ²⁰⁰¹). Cysteine serves as a precursor for a range of further reduced sulfur‐containing compounds, such as methionine, S‐adenosylmethionine, glucosinolates, and GSH (Lunde et al. ²⁰⁰⁸). In addition, sulfur is important in formation of the iron–sulfur cluster in the photosynthetic apparatus and in the electron transport system that acts as a major sink for Fe and are essential for many cellular enzymatic reactions. Fe–S clusters are found in the structure of cytochrome b6f, ferredoxin, and PSI reaction centers (Allen 2004), and sulfur deprivation has been shown to cause significant suppression of photosynthetic efficiency (Resurreccion et al. ²⁰⁰²). Indeed, lack of sulfur has direct effects on photosynthesis via a reduction in chlorophyll synthesis. In addition, many enzymes involved in photosynthesis, such as ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO), require sulfur for correct functioning. Therefore, sulfur deprivation can inhibit activity of these enzymes, reducing plant ability to fix carbon. Cellular levels of reactive oxygen species (ROS) are also associated with sulfur availability. This is because sulfur is essential to produce antioxidant compounds (e.g., glutathione), and poor photosynthetic efficiency leads to inefficient use of sunlight, which leads to further accumulation of ROS. These molecules damage cell membranes and organelles, including chloroplasts, further compromising photosynthesis (Lencioni et al. ¹⁹⁹⁷; Leustek & Saito ¹⁹⁹⁹). Previous experiments have proved that S fertilization improves photosynthesis and growth through regulation of N assimilation (Rais et al. ²⁰¹³). These authors found that the two nutrients work coordinately in enhancing N assimilation, resulting in prevention of chlorophyll degradation, and increased photosynthetic efficiency and growth.

Light, apart from serving as the energy source for photosynthesis, acts as a morphogenetic signal and affects leaf, stomatal, and chloroplast movement (Goto 2003). Light also induces synthesis of protective metabolites, including anthocyanins (Kami et al. ²⁰¹⁰). These responses are directly influenced by light quality, intensity, and photoperiod (Kang et al. ²⁰¹³). Alterations in the light environment trigger multiple photoreceptors that affect plant growth as through the integration of multiple signal transduction pathways (Goto 2003) in which water uptake and transport are involved (Kenefick et al. ²⁰⁰²). Although a complex interaction between light and temperature has been reported (Gray et al. ¹⁹⁹⁷; Li et al. ²⁰²²), Willmer & Fricker (1996) demonstrated that low light is particularly effective in regulating stomata. Therefore, the fact that light drives the response of guard cells independent of chloroplast‐driven changes to CO₂ is an important issue (Makino & M. ¹⁹⁹⁹). As the effect of light intensity has been reported to regulate water homeostasis (Wang et al. ²⁰²⁴), the different parameters that determine water status, such as osmotic potential (Ψπ) and water potential (Ψw) (Turner 1981), should be related to chlorophyll fluorescence measures, such as Fv/Fo and Fv/Fm. These parameters are key indicators of photosynthetic status and are closely associated with the efficiency of photosystem II (PSII) (Krause & Weis ¹⁹⁸⁴). Accordingly, as plants respond to light stress by regulating water homeostasis (Ding et al. ²⁰¹⁹), investigating the role of aquaporins (AQPs) is also essential, since these membrane proteins dynamically optimize plant hydraulic properties (Wang et al. ²⁰²⁴). Based on the published literature, there is a direct relationship between PIP aquaporins transcript levels, leaf hydraulic conductance, and light exposure (Cochard et al. ²⁰⁰⁷). Additionally, the close relationship between aquaporins, nutrients, and CO₂ transport make these transmembrane channels essential for adequate nutrient distribution and optimal photosynthetic activity (Barzana et al. ²⁰²¹; Lopez‐Zaplana et al. ²⁰²²).

Aquaporins are membrane proteins belonging to the major intrinsic protein (MIP) superfamily (Kruse et al. ²⁰⁰⁶). They often form tetrameric structures, functioning as water transport channels in biological membranes. Water uptake by roots occurs through sequential transport along radial and axial pathways. Axial transport is mediated by xylem vessels, which lack significant membrane barriers, while radial transport involves three concurrent pathways: apoplastic (through cell walls), symplastic (via plasmodesmata and cytoplasmic connections), and transcellular (across membranes). Many aquaporins are highly expressed in roots, indicating their critical role in root water transport (Li et al. ²⁰¹⁴). Higher plant aquaporins fall into five subfamilies, depending on their sequence homology and subcellular location. Three of these, the plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), and nodulin 26‐like intrinsic proteins (NIPs), are well‐described with respect to protein localization and function. Two additional subfamilies, small basic intrinsic proteins (SIPs) and uncategorized intrinsic proteins (XIPs), were discovered more recently (Maurel et al. ²⁰¹⁵). Recently, 64 aquaporins have been identified in broccoli (Nicolas‐Espinosa & Carvajal ²⁰²²) so their individual study is required.

The joint effect of light and sulfate deficiency on broccoli development is unknown, especially regarding aquaporin expression. In this context, this study analysed the effects of different light intensities and sulfur deficiency on the physiological development of broccoli. For this, water relations, mineral composition, and glucosinolate concentration in relation to expression of PIP1.2 and PIP2.7 aquaporins was examined. The selection of these two aquaporins is based on previous studies and molecular functions (Nicolas‐Espinosa et al. ²⁰²⁴).

MATERIAL AND METHODS

Plant growth conditions

Broccoli seeds (Brassica oleracea var. italica cv. Parthenon) were hydrated for 24 h in distilled water under constant aeration. They were then transferred for 48 h to a tray containing vermiculite for germination in complete darkness at 28°C. Subsequently, germinated seeds were placed in a controlled environment chamber with a photoperiod of 16 h light/8 h darkness, at 24/20°C, relative humidity of 60%, for an additional 48 h. After this, the sprouts were transferred to a hydroponic cultivation system on Hoagland solution, with continuous aeration for 1 week. Four separate hydroponic tanks were employed, each containing nine plants. Two tanks were exposed to photosynthetically active radiation (PAR) of 400 μmol m⁻² s⁻¹ designated as control light intensity treatment (C). The remaining two tanks were exposed to PAR of 200 μmol m⁻² s⁻¹, referred to as low light treatment (L). After 1 week of growth, sulfur deprivation was induced by omitting sulfur (MgSO₄) from the Hoagland solution in one of the two tanks for each light condition, resulting in sulfur‐deficient control tanks (‐S) and sulfur‐deficient low‐intensity tanks (L‐S). To retain the Mg concentration comparable between the two treatments, 0.825 mM MgCl₂ was added the ‐S tanks. The sulfur deprivation treatment was maintained for 11 days, after which plants were harvested and separated into aerial and root tissues. Four plants from each treatment group (C, ‐S, L, L‐S) were immediately stored at – 80°C for subsequent analysis, while the remaining plants were used for additional measurements.

Physiological measurements

After harvesting remaining plants from the tanks, fresh weight (FW) and root length were measured, along with leaf area and leaf weight for each treatment. Leaf area was measured using ImageJ (Schneider et al. ²⁰¹²). Later, samples were transferred to an oven (60°C) and dried to complete dehydration. Dry samples were weighed for evaluation of shoot, root, and leaf dry weight (DW). Leaf data were used to obtain leaf mass per area (LMA) using the following formula:

$$\mathrm{LMA}=\mathrm{Dry}\text{mass of the leaf}\left(\mathrm{g}\right)/\text{Leaf area}\left({\mathrm{m}}^2\right)$$

Measurements of stem height and petiole length were performed manually on days 2, 4, 8, and 11 after application of sulfur treatment.

Osmotic potential (Ψμ) of three plants per treatment was measured using a freezing‐point depression osmometer (Digital Osmometer, Roebling, Germany) at 25 ± 1°C (Navarro et al. ²⁰⁰³).

Chlorophyll fluorescence measurements (Fv/Fm–Fv/Fo) were taken using a chlorophyll fluorimeter (OS‐30p + Chlorophyll Fluorimeter, OPTI‐SCIENCES). Five plants were measured for each treatment. Measures were taken on suitable leaves after 30 min of dark adaptation on days 2, 8, and 11 in the middle of the light period.

Water parameters

The leaf water potential (Ψω) was measured on the fourth fully expanded leaf of five plants from each treatment (C, ‐S, L, L‐S) using a Scholander pressure chamber (Skye Instruments, UK) (Turner 1988). Turgor potential (Ψp) was calculated as the difference between leaf water potential and osmotic potential (Nonami & Schulze ¹⁹⁸⁹).

Relative water content (RWC) was determined using leaf discs collected at harvest to minimize the need for entire plant sampling. Fresh weight (FW) was recorded immediately after harvesting the discs, while turgid weight (TW) was measured following 24 h immersion in de‐ionized water. Dry weight (DW) was obtained after drying discs for 48 h in an oven at 60°C. RWC was calculated using the following equation:

$$\mathrm{RWC}\left(\%\right)=\begin{array}{l}\left[\left(\text{Fresh Weight}-\mathrm{Dry}\text{Weight}\right)/\left(\text{Turgid Weight}-\mathrm{Dry}\text{Weight}\right)\right]\times 100\end{array}$$

while humidity:

$$\text{Humidity}\left(\%\right)=\begin{array}{l}\left[\left(\text{Fresh Weight}-\mathrm{Dry}\text{Weight}\right)/\text{(Fresh Weight)}\right]\times 100\end{array}$$

Glucosinolates analysis

Glucosinolates were isolated from leaves of three plants per treatment, following the protocol described by Albaladejo‐Marico, Yepes‐Molina et al. (2024). Samples were analysed using high‐performance liquid chromatography–mass spectrometry (HPLC‐MS) following the procedure described by Albaladejo‐Marico, Carvajal et al. (2024). This was carried out in negative ion mode on an Agilent 1290 Series II HPLC connected to an Agilent iFunnel 6550 quadrupole time‐of‐flight (Q‐TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent Jet Stream (AJS) Dual Electrospray Ionization (ESI) system.

Analysis of mineral nutrients

Concentrations of macro‐ and micronutrients were quantified in samples of the dried aerial tissues and roots of three individual plants from each treatment. These samples were finely ground into homogeneous powder. Analyses used Inductively Coupled Plasma‐Optical Emission Spectrometry (ICP‐OES) on a Thermo ICAP 6500 Duo instrument (Thermo Fisher Scientific, Waltham, MA, USA) as described in Nicolas‐Espinosa et al. (2024). Nutrient concentrations were expressed as mg 100 g⁻¹ DW for macronutrients and mg g⁻¹ DW for micronutrients.

RNA extraction and quantitative real‐time PCR (RT‐qPCR)

Frozen aerial tissues and roots from four replicates per treatment were pulverized into a fine powder in liquid nitrogen. Total RNA was extracted from 100 mg powdered material utilizing the NZY Plant/Fungi RNA Isolation Kit (Nzytech, Lisbon, Portugal) according to the manufacturer's protocol. RNA purity and concentration were assessed via a Nanodrop 1000 spectrophotometer (ThermoFisher Scientific). Extracted RNA samples were preserved at −80°C until further analysis. The RT Master Mix for qPCR II Kit (HY‐K0510A‐MedChemExpress, Sollentuna, Sweden) was used to synthesize cDNA from 2 μg total RNA, according to the manufacturer's protocol.

Quantitative real‐time PCR (RT‐qPCR) was performed to analyse expression of two aquaporin genes (PIP1.2, PIP2.7) using gene‐specific primers designed as described in Nicolas‐Espinosa et al. (2024) on an Applied Biosystems 7500 Real‐Time PCR system utilizing 2 μL cDNA samples (1:10 diluted) within an 8 μL reaction mixture (600 nM specific primers, 5 μL SYBR Green Master Mix 2X (Applied Biosystems) and nuclease‐free water) using an Applied Biosystems 7500 Real‐Time PCR system. The amplification protocol involved two steps: initial denaturation at 95°C for 10 min, followed by 40 cycles of 15 s denaturation at 95°C, and 1 min annealing and extension at 60°C. Subsequently, a dissociation stage was performed. These conditions were used for both target and reference genes. Three technical replicates and three biological samples were tested for each treatment. Transcript levels were calculated using the 2^−ΔΔCt method (Livak & Schmittgen ²⁰⁰¹) for both target and reference genes.

Statistical analysis

Statistical analyses and data presentation used Origin (Pro) (v. 2021 software; OriginLab, Northampton, MA, USA). Significant differences among values of all the parameters were determined at P ≤ 0.05, according to Tukey's test. Significant changes produced by single factors (light and sulfur) and interaction between factors for physiological values, mineral composition, and AQP expression were studied using a two‐way ANOVA (Table S1).

RESULTS

Plant growth

Analysis of stem height data showed no changes during the first 8 days after the start of the sulfur deficiency treatment. On day 11, the L treatment had the tallest stems, while the control was shortest (Fig. 1A). There were differences in petiole length between control and light treatment from day 2, where the control had the highest value compared with other treatments, and largest differences on day 11 (Fig. 1B). The interaction between light and sulfur was not significant, and the L‐S treatment result was a combination of the single factors (Table S1).

Fig. 1.

Physiological measures in C, C‐L, L and L‐S plants. (A) Anatomical differences. (A) Stem height (cm) on days 2, 4, 8, and 11. (B) Petiole length (cm) on days 2, 4, 8, and 11. (C) Total fresh weight (g). (D) Total dry weight (g). (E) Root fresh weight (g). (F) Root dry weight (g). (G) Root length (cm). Different letters indicate significant differences according to ANOVA followed by post hoc Tukey test (P < 0.05). Two‐way ANOVAs in panels (A) and (B) were performed within a day of measurement. Each bar represents mean ± SE (n = 6).

Plants subjected to sulfur deficiency (‐S) and light stress (L) had a significant reduction in total FW compared to the controls. The combination of light stress and sulfur deficiency (L‐S) resulted in the lowest FW, as expected (Fig. 1C). There was a similar trend in root FW, with a significant decrease across treatments, except for the ‐S treatment, which did not differ from the control (Fig. 1E). Patterns were comparable for DW at root level. At shoot level, DW was significantly reduced only under L and L‐S treatments, with no change under S deficiency. There were no significant differences between the two control treatments, while L and L‐S results were significantly lower for both total and root DW (Fig. 1D,F). The longest roots were in the low‐intensity treatment, while only the L‐S treatment was significantly different from the C treatment (Fig. 1G). Only total DW showed a significant interaction between light and S deficiency (Table S1).

Only in treatment ‐S was leaf humidity significantly lower, suggesting that sulfur deficiency might affect plant water relations (Fig. 2A). Regarding LMA, the highest was in the ‐S treatment (Fig. 2B). The two light treatments had the lowest values, compared to the C treatment, indicating that only light treatment negatively affects the LMA by decreasing DW. For RWC, only L‐S treatment was significantly lower (Fig. 2C).

Fig. 2.

Water relations in C, ‐S, L, and L‐S treated plants. (A) Humidity. (B) Leaf mass area. (C) Relative water content (%). Different letters indicate significant differences according to ANOVA followed by post hoc Tukey test (P < 0.05). Each bar represents mean ± SE (n = 5).

Water, osmotic, and turgor potentials are provided in Table 1. Control plants had a higher water potential while all other treatments had more negative values. There were no differences in water potential between ‐S, L, and L‐S. However, for osmotic potential, only the ‐S treatment was significantly higher, explaining why turgor was also highest in this treatment. There were no significant differences in the other treatments.

Table 1.

Water potential (Ψw), osmotic potential (Ψπ), and turgor potential (Ψp) in treated plants.

Treatment	Ψw	Ψπ	Ψp
C	−0.130 ± 0.008 a	−2.52 ± 0.13 ab	2.39 ± 0.14 ab
‐S	−0.296 ± 0.005 b	−3.27 ± 0.35 b	2.97 ± 0.34 a
L	−0.312 ± 0.005 b	−2.16 ± 0.12 a	1.85 ± 0.12 b
L‐S	−0.318 ± 0.004 b	−2.44 ± 0.10 a	2.13 ± 0.10 b

Different letters indicate significant differences according to ANOVA followed by post hoc Tukey test (P < 0.05). Values are mean ± SE (n = 5).

The Fv/Fo is as an indicator of photosystem II (PSII) efficiency. There was a reduction in this ratio on each sampling day, with the light treatment plants having the lowest value in the L‐S treatment (Fig. 3A). There was no effect of sulfur (‐S) alone on Fv/Fm, whereas both L and L‐S had reduced Fv/Fm on day 2 (Fig. 3B). On days 8 and 11, plants under low light (L) recovered Fv/Fm to control levels. However, there was no recovery under combined light and sulfur stress (L‐S), indicating an interaction between the two.

Fig. 3.

Chlorophyll fluorescence measurements on broccoli grown under C, C‐S, L and L‐S on days 2, 8, and 11. (A) Fv/Fo on days 2, 8, and 11. (B) Fv/Fm on days 2, 8 and 11. Different letters indicate significant differences according to ANOVA followed by post hoc Tukey test (P < 0.05). Each bar represents mean ± SE (n = 5).

Mineral composition differed between treatments in both aerial and root tissues. In particular, reductions in N, S, and Mg in aerial tissue of sulfur deficient plants (Fig. 4A,B,E). In roots, only N decreased in S plants and Mg in L‐S plants. K did not change in leaves or roots (Fig. 4C). P decreased only in root tissues under S deficiency (Fig. 4D). Zn concentration did not change in aerial tissue but increased in root tissue under both single and combined stress treatments (Fig. 6F). B increased in aerial tissues only under low light but decreased in roots in the L‐S treatment (Fig. 4G). Mo significantly increased under sulfur deficiency in both aerial and root tissues, as well as under L‐S treatment (Fig. 6H). Only Mg, Zn, B, and Mo showed a significant interaction between light and sulfur (Table S1).

Fig. 4.

Mineral composition of treated aerial and root tissue. Concentration of N (A), S (B), K (C), P (D), Mg (E), Zn (F), B (G), and Mo (H). Different letters indicate significant differences according to ANOVA followed by post hoc Tukey's test (P < 0.05). Each bar represents mean ± SE (n = 3).

Fig. 6.

Leaf glucosinolates concentrations. (A) Glucobrassicin concentration (μg g⁻¹ FW) in C, ‐S, L, and L‐S plants. (B) Glucoraphanin concentration (μg g⁻¹ FW) in C, ‐S, L, and L‐S plants. Different letters indicate significant differences according to ANOVA followed by post hoc Tukey test (P < 0.05). Each bar represents mean ± SE (n = 3).

Principal Components Analysis (PCA) of aerial tissue revealed a strong association among all sulfur‐deficient plants, closely linked to molybdenum (Mo) concentrations (Fig. 5A). In aerial tissues there was a clear separation between treatments, except for L‐S and ‐S plants, which clustered. In contrast, root tissues showed greater dispersion with weaker interactions between treatments: no significant separation between roots of C and L plants. Interestingly, there was a notable association in L‐S roots with Mo concentration (Fig. 5B). In data for whole plants there were four distinct groups (Fig. 5C). Sulfur‐deficient plants were clearly correlated with Mo concentration, with an even stronger association in L‐S‐treated plants. Control roots were associated with B and Mg, while L plants had similar associations with B and Mg in aerial tissues.

Fig. 5.

PCA of plant mineral composition of micro‐ and macronutrients. Arrows indicate loadings of each nutrient; 95% confidence ellipses plotted for combined tissue samples, each small circle represents data from an individual sample, while a large circle represents centroid of mean value. (A) PCA using measures from aerial part of treated plants. (B) PCA using measures from root of treated plants. (C) PCA using both aerial and root measures of treated plants (n = 3).

The concentration of glucobrassicin in C and L treatments was ca. 300 μg g⁻¹ and 1.2 μg g⁻¹ for glucoraphanin. These concentrations declined significantly under sulfur deficiency, reaching almost undetectable levels in both glucosinolates measured. An exception was glucobrassicin in the L‐S deficiency treatment, where the value was three times lower the control (Fig. 6A).

Expression of the selected aquaporins differed between treatments and plant tissues. In leaves, PIP1.2 was five times less expressed in the ‐S and L treatments, although there were no changes in L‐S treatment expression (Fig. 7A). PIP2.7 in leaves was almost four times more in the L‐S treatment compared with control treatment, with no significant differences between control ‐S and L (Fig. 7B). However, in root tissue PIP1.2 was less expressed in all treatments, with lowest expression in the L‐S treatment (Fig. 7C). A similar pattern was observed for PIP 2.7 as its expression was reduced in all treatments (Fig. 7D).

Fig. 7.

Gene expression of different aquaporins expressed as fold change with respect to control. (A) PIP 1.2 relative expression in aerial tissue in C, ‐S, L, and L‐S plants. (B) PIP 2.7 relative expression in aerial tissue in C, ‐S, L, and L‐S plants. (C) PIP 1.2 relative expression in root tissue in C, ‐S, L, and L‐S plants. (D) PIP 2.7 relative expression in root tissue in C, ‐S, L, and L‐S plants. Different letters indicate significant differences according to ANOVA followed by post hoc Tukey test (P < 0.05). Each bar represents mean ± SE (n = 3).

DISCUSSION

Light and nutrients are abiotic factors that directly affect plant growth rate. The light conditions used in this experiment correspond to those on cloudy days or to shading caused by weeds, while the simulated sulfur shortage can be found in poor quality soils or in plant–plant competition. Exposure to these environmental stresses triggers different responses. Regarding stem height, there were significant differences only 11 days after applying treatments and between the C and L treatments, but not ‐S treatment. Therefore, low light intensity was the determining factor that triggered changes in plant height. This is consistent with the reduced petiole length results, in favour of stem elongation, only under low light (alone or in combination with sulfur shortage) (Fig. 1). Light intensity can affect plant growth even during early germination stages (Gao et al. ²⁰²¹), and low light levels can increase plant height (Frąszczak et al. ²⁰⁰⁸). This is confirmed by the longer stem in the L treatment to increase efficiency of light capture under shade and/or low light (Ma & Li ²⁰¹⁹). As the increase in stem height was less pronounced in the L‐S treatment, it is likely that combined stress prevented adaptation to light limitation due to the metabolic disruptions induced by sulfur deficiency. Under sulfur shortage, synthesis of sulfur‐containing metabolites is impaired, affecting plant morphology and total biomass partitioning (Houhou et al. ²⁰¹⁸).

Variations in plant biomass revealed that stress decreased growth rate, which consequently reduced both FW and DW. Light had a larger effect on weight than sulfur treatments, but the combined treatments resulted in the most significant weight loss. Olesen & Grevsen (1997) reported dry mass reduction in plants exposed to high irradiance, with similar results under shade (Ampong‐Nyarko et al. ¹⁹⁹²; Gibson et al. ²⁰⁰¹), indicating the strong influence of light level on DW. The role of sulfur in plant development is well documented in Brassica (Scherer 2001; Malhi et al. ²⁰⁰⁵) and in other important crops, e.g., corn (Huang et al. ²⁰¹⁸). There was no correlation between FW and DW in the ‐S treatment as a consequence of reduced leaf water content compared to control plants (Fig. 1C,D). This is a possible adaptation of leaf morphology to sulfur deficiency to avoid water loss that is not found under low light. Kastori et al. (2000) reported that interruption of S supply significantly reduced stomatal density and increased stomatal diffusive resistance, both of which are typical responses to avoid water loss. These results are consistent with reduced FW and leaf water potential, unchanged DW and photosynthetic efficiency, and increased LMA observed in sulfur deficient plants (Figs. 1C,D, 2, and 3) (Baird et al. ²⁰¹⁷). Light and sulfur can therefore be considered regulators of plant water dynamics when they are insufficient/suboptimal for normal growth and development.

Root morphology changed, with longer roots in L‐S plants corresponding to very low FW and DW. Sulfur deprivation can modify root architecture of Brassica napus (Weese et al. ²⁰¹⁵) and increase root length in wheat (Carciochi et al. ²⁰¹⁷). Alterations in root morphology in response to nutrient deficiencies have been documented for several essential elements, including P (Niu et al. ²⁰¹³), N (Ötvös et al. ²⁰²¹), and K (Sustr et al. ²⁰¹⁹), highlighting their crucial roles in root development.

The relative water content (RWC) was generally low, likely indicating mild stress in plants during measurements and previously reported for other Brassica species (Rhythm & Sardana ²⁰²²). Notably, only the ‐S treatment had a significantly lower RWC than control plants, further supporting the presence of water stress or water‐related physiological disturbances in response to sulfur deficiency. Sulfur nutrition has an important role in response to water deficit (Henriet et al. ²⁰¹⁹), where sulfur concentration increases in xylem sap and triggers stomatal closure through activation of genes involved in abscisic acid (ABA) synthesis. Similarly, certain rapeseed cultivars with constitutively higher sulfur uptake had enhanced tolerance to water stress (Lee et al. ²⁰¹⁶). As observed here in hypersensitivity of sulfur‐deficient plants to moderate water deficits, reinforcing the critical role of sulfur in water stress resilience.

Plant total DW was only reduced under low light, alone or in combination with the S treatment (Fig. 1D). This is consistent with reduced photosynthetic efficiency, as demonstrated by the lower Fv/Fm in L and L‐S treated plants on day 2 (Fig. 3B). Particularly relevant is response to these stressors on days 9 and 11, when L plants were able to adapt to low light (L), but not the L‐S plants. The relationship between light intensity and dry matter accumulation is correlated with photosynthetic activity (Lichtenthaler et al. ¹⁹⁸¹). However, the combined effect of both stresses in L‐S treatment is likely associated with significant chlorophyll reduction in response to S deprivation, as reported in earlier (Astolfi et al. ²⁰⁰¹).

The reduction in N concentrations in plants exposed to sulfur deficiency is similar to results of Rais et al. (2013), who proposed that adequate sulfur supply is necessary for good N assimilation. The increase in Zn concentrations in all treatments may be attributed to its essential role in enzyme activity, as plants experiencing stress can activate enzymatic pathways that require Zn (Gupta et al. ²⁰¹⁶).

Regarding Mo, its main function is in N and S metabolism. There was an increase of Mo concentrations in S‐deficient plants, similar to results of previous studies (Alhendawi et al. ²⁰⁰⁵). As both N and S uptake were reduced, an increase in Mo might be an attempt by the plant to increase N and S assimilation from the medium, as Mo regulates N‐assimilation enzyme activity and expression (Imran et al. ²⁰¹⁹). Mo is also an essential micronutrient with an important role in photosynthesis because of its involvement in chlorophyll biosynthesis, configuration, and ultrastructure (Imran et al. ²⁰¹⁹). The role of Mo in the photosynthetic machinery has been studied in Brassica napus where a Mo concentration of 0.15 mg kg⁻¹ increased all photosynthetic parameters (Qin et al. ²⁰¹⁷). These Mo values are similar to those in our sulfur deficiency treatments, indicating another possible response involving Mo to assure growth and tolerance to ongoing stresses.

The above response could also be linked with the role of Mo in plant defence as it is a cofactor for different enzymes of plant antioxidant machinery (Hayyawi et al. ²⁰²⁰). By reducing accumulation of glucosinolates, which are important molecules for both biotic and abiotic stress tolerance (Del Carmen Martínez‐Ballesta et al. ²⁰¹³), sulfur deficiency could have impaired the defence mechanism in broccoli. Therefore, increased Mo leaf concentrations could represent a response to the inhibited synthesis of glucosinolates, possibly compensating for the reduced defence caused by sulfur deficiency. The PCA results indicated distinct nutrient associations. In aerial tissues, sulfur‐deficient plants show a strong association with Mo concentration. In roots, sulfur‐deficient plants were associated with Mo and Zn. Combining the data, sulfur‐deficient plants remain linked to Mo, supporting Mo as a nutrient with regulatory functions in photosynthesis, as S deficiency reduces chlorophyll biosynthesis (Kumawat et al. ²⁰⁰⁶). Control plants exhibited relationships with B and Mg in roots, while L plants showed similar associations with B and Mg in aerial tissues. B concentration remained low in roots, indicating its major role in the shoot where, under stress, light affects Mg levels, which are involved in photosynthesis (Mitra 2018).

Several possible mechanisms underlying similar nutrient imbalance have been previously described (Mcgrath & Lobell ²⁰¹³). Elevated atmospheric CO₂ led to a decline in concentrations of several mineral nutrients, including Mg and N, while increasing Mo levels, a pattern that closely mirrors the mineral profile alterations observed here under sulfur deficiency. Under elevated CO₂, reductions in transpiration and shifts in internal physiological nutrient demand were identified as primary factors influencing mineral composition, alongside decreased nutrient flow to roots. Similarly, sulfur deficiency may trigger comparable physiological responses. Sulfur deficiency is linked to a reduction in photosynthetic activity, which, in conjunction with impaired water status and decreased stomatal density, likely contributes to a decline in transpiration rate. These physiological changes may underlie the observed alterations in mineral nutrient concentrations, as reduced water movement through the plant constrain both nutrient uptake and internal nutrient transport. Furthermore, nutrient remobilization may be activated, particularly for elements such as Mg, which plays a central role in photosynthesis (Hauer‐Jákli & Tränkner ²⁰¹⁹). As photosynthetic activity is suppressed under sulfur deficiency, the demand for Mg is correspondingly reduced, supporting its redistribution or downregulation.

With respect to glucosinolates, levels were the same in control and L treatment, suggesting that synthesis of these secondary metabolites does not depend on light availability but on sulfate bioavailability. As is well established, glucosinolate biosynthesis begins at early stages of plant growth and persists throughout all development stages (Guo et al. ²⁰¹³; Albaladejo‐Marico, Carvajal et al. ²⁰²⁴; Albaladejo‐Marico, Yepes‐Molina et al. ²⁰²⁴). Glucosinolates are characterized by a basic structure with a thioglucose moiety, a sulfonated oxime, and a side chain derived from amino acids (Halkier & Du ¹⁹⁹⁷), where sulfur is an essential component. Hence, the catabolic metabolism of glucosinolates as a sulfur reservoir has been recently investigated, revealing potential metabolic mechanisms involved in this process (Sugiyama et al. ²⁰²¹). The present findings suggest that plants experiencing sulfur deficiency actively utilize glucosinolates as sulfur reservoirs. In sulfur‐deficient plants, available glucosinolates are minimal, rendering them undetectable. Conversely, L‐S plants exhibit enhanced regulation of glucosinolates levels, likely attributable to reduced metabolism, as indicated in FW and DW values. This reduction in metabolic activity is primarily associated with reduced photosynthetic efficiency because of sulfur deprivation, which is further exacerbated under low light. The decreased growth rate in L‐S plants may lead to slower utilization of glucosinolates as sulfur reservoirs, potentially explaining the concentrations detected.

The expression profile of the selected aquaporins, PIP1.2 and PIP2.7, in the L treatment in leaf tissue revealed a direct relationship between aquaporin expression and light deficiency, as found previously (Cochard et al. ²⁰⁰⁷; Kim & Steudle ²⁰⁰⁹). The same was observed in ‐S leaf tissue. The significant reduction in N in the sulfur‐deficient plants could also explain the observed reduced aquaporin expression. N assimilation is very closely related to aquaporin expression (Ishikawa‐Sakurai et al. ²⁰¹⁴; Gao et al. ²⁰¹⁸), especially with respect to root water uptake (Ren et al. ²⁰¹⁵). It is also essential to compare expression levels with corresponding osmotic potentials. Only the ‐S treatment resulted in a reduction in osmotic potential compared to the control. This suggests that water regulation under sulfur deprivation is not solely dependent on aquaporin expression but also involves accumulation of osmolytes, as a lower osmotic potential facilitates water uptake. These changes in osmotic potential were not seen in the L treatment, indicating a different water regulation route but the same aquaporin expression reduction.

Our results support formulation of a hypothesis regarding the physiological mechanisms by which sulfur deficiency altered plant water status. Narayan et al. (2023) reported a decrease in root hydraulic conductivity under sulfur deficiency, suggesting disrupted water uptake and transport processes associated with nutrient starvation signalling pathways. When coupled with the observed downregulation of aquaporin gene expression in root tissues, our findings collectively indicate a compromised water transport system, confirming a disturbed water balance under sulfur deficiency. In response to such impaired water availability, plants should initiate adaptation mechanisms. One of the most prominent is stomatal closure, a strategy to reduce transpirational water loss. This response is tightly regulated by biosynthesis of ABA, is directly dependent on cysteine metabolism, and reported to be influenced by sulfur availability (Bouranis et al. ²⁰²⁰). Consequently, sulfur‐deficient plants are forced to increase stomatal diffusive resistance and reduce stomatal density, promoting effective stomatal closure as a means of water conservation (Kastori et al. ²⁰⁰⁰). This physiological adjustment may also account for altered nutrient concentrations, as a decrease in transpiration limits root nutrient uptake and transport through the xylem.

In our study, only plants subjected to sulfur deficiency (S treatment) exhibited water‐related physiological constraints, leading to morphological and physiological adaptations aimed at mitigating stress. The combined stress exacerbated decreases in other physiological parameters, such as biomass accumulation, photosynthesis, and mineral concentration, indicating a more severe stress state, but there were no indicators of water imbalance. This can be explained by examining aquaporin expression patterns in L‐S plants. Expression of the two leaf aquaporins is the same as in the control in the case of PIP1.2, and highly increased in the case of PIP2.7, showing how the plant tries to increase or maintain PIP expression to maintain plant water homeostasis. Nicolas‐Espinosa et al. (2024) reported that B deficiency reduced expression of PIP1.2 and PIP2.7. However, when combined with an additional abiotic stress, such as salinity, expression either increased or returned to control levels, as also observed in our study. These results clearly show how combined exposure to these stresses trigger different regulation mechanisms in leaf aquaporins, which are necessary for plant survival. In roots, differences in aquaporin expression between treatments were less pronounced, but there was a general downregulation across all treatments for both aquaporins. Under these conditions, the L‐S treatment did not exhibit a distinct expression pattern, suggesting that the proposed alternative regulatory mechanism for aquaporins under combined stress is exclusively active in leaf tissue, highlighting its functional significance in water regulation in this organ.

CONCLUSION

This study demonstrates how exposure to light stress and sulfur deficiency modulate key plant responses to enhance adaptation through regulation of water dynamics. A conceptual diagram summarizing our findings is presented in Fig. 8. Sulfur deficiency had a more pronounced impact on plant water relations than light stress, triggering both physiological and morphological adjustments to maintain water homeostasis. In contrast, light stress primarily influenced photosynthetic activity, directly reducing biomass accumulation. Notably, plant responses were significantly altered and more pronounced under combined stress conditions compared to individual treatments. A particularly striking observation was the differential regulation of aquaporin expression under combined stress, diverging from the patterns observed under single‐stress conditions, suggesting a critical compensatory mechanism aimed at preserving optimal water balance within the plant.

Fig. 8.

Conceptual diagram summarizing the main results. Left: representation of experimental design and description of general effects in the single stress treatments, highlighting the reduction in aquaporin expression affecting nutrient and water transport. Right: description of general effects observed in combined stress treatment, highlighting unique responses, such as reduced metabolic activity and increase in aquaporin expression as mechanisms to assure plant survival.

AUTHOR CONTRIBUTIONS

MCA, AM, VC, and JNE contributed to conception and design. AAL and OP carried out the experiments. AAL prepared figures and tables, and prepared the first draft of the manuscript. MCA, AAA, AAA, JNE, and OPP contributed to manuscript revisions, read and approved the submitted version. MCA obtained the funding. All authors have read and approved the manuscript.

FUNDING INFORMATION

This research was funded by Spanish Ministerio de Ciencia e Innovación (CPP2022‐009860).

Supporting information

Table S1. The interactions between low light and sulfur deficiency using two‐way ANOVA on different measured variables. Significant differences in all the parameters were determined at P ≤ 0.05, according to Tukey's test.