Z. mays seeds (cv. Bilicious) were purchased from Halifax Seed (Halifax, NS, Canada). ANE, commercially available as Acadian Marine Plant Extract (Acadian Seaplants Ltd., Dartmouth, NS, Canada), was used as the seaweed biostimulant. The chemical composition of the extract was described by Rayirath et al. (2009). The P content in the 0.01% of ANE was measured using inductively coupled plasma optical emission spectrometer (ICP-OES) at the Analytical and Dairy Lab, Harlow Institute, Nova Scotia, Canada.

Seeds were sterilized using 2.5% Clorox® and were kept on filter paper moistened with water in a petri dish for germination in the dark. Seven-day-old, uniformly germinated, seedlings were used for further experiments. The experiment was designed to assess the effects of 0.01% ANE on the growth of 7-day-old Z. mays seedlings grown under P-limited conditions for 14 days. This concentration of ANE was selected based on the results reported in previous studies (Shukla et al., 2018; Bajpai et al., 2019; Jithesh et al., 2019). In addition to the control (1/2 MS (Murashige and Skoog media), C), the other three treatments were 1/2 MS + 0.01% ANE (T₁), 1/2 MS-P (T₂), and 1/2 MS-P + 0.01% ANE (T₃). The MS medium without P (MSP19) was procured from Caisson Labs, USA. It was estimated that 0.01% ANE contained 5.0 μM P, which was added to T₂ in the form of H₃PO₄ (Rickard, 2000). The plants treated with different treatments were grown for 14 days in the growth chamber maintained at 24°C with a photoperiod (100 μmol photons/m²/s) of 16/8 h (day/night). Increase in total root length and leaf area of plants was recorded at 14 days after the imposition of P-limited conditions by using ImageJ software. After 14 days under different conditions, fresh weights of shoots and roots were recorded. DW was recorded after drying in an oven at 70°C for 72 h. The data for the morphological parameters were generated using six plants for each experimental condition, and the experiment was triplicated.

The nitrogen content and mineral element analysis were done at the Analytical and Dairy Lab, Harlow Institute, Nova Scotia, Canada. The plants grown under different treatments for 14 days were dried at 72°C for 72 h. The nitrogen content in the plant tissue was determined by combustion analysis method using Leco CN828 Carbon Nitrogen Determinator. The mineral elements were measured from the ash dried plant samples with an ICP-OES.

The effect of ANE on chlorophyll a and b and the carotenoid content of Z. mays grown under different treatments for 14 days were determined according to the protocol described by Lichtenthaler (1987). Briefly, 1 g of six individual plant samples was instantaneously ground using a mortar and pestle in 5 ml of cold methanol. Following extraction, the ground mixture was centrifuged at 10,000 rpm at 4°C for 10 min, and the pellet was re-extracted with 10 ml of cold methanol until all color was removed. Two extracts were combined, and total volume was made up to 15 ml. Absorbance was measured at 470, 652.4, and 665.2 nm using a UV–VIS spectrophotometer (Biotek, USA). The chlorophyll contents were calculated according to Lichtenthaler (1987).

Chl_a = 16.72 A_665.2 – 9.16 A_652.4

Chl_b = 34.09 A_652.4 – 15.28 A_665.2

Carotenoids = (1,000 A₄₇₀ – 1.63 Chl_a – 104.96 Chl_b) / 221

The effect of ANE on the anthocyanin content of Z. mays grown under different treatments for 14 days was evaluated using the protocol published by Burgos et al. (2013). One gram of plant sample was extracted with 10 ml of methanol and acidified with 1% HCl. The mixture was centrifuged at 5,000 rpm for 10 min at 4°C, and the pellet was re-extracted. Different fractions were combined, and the final extract volume was made up to 25 ml. Absorbance at 545 nm was recorded using a UV–VIS spectrophotometer. The anthocyanin content was calculated using the molar extinction coefficient and molecular weight of malvidin-3-p-coumaroyl-glucoside (i.e., 545 nm, 3.02 × 104 L/mol/cm, 718.5 g/mol).

Electrolyte leakage was determined as described by Shukla et al. (2012). The youngest, fully expanded leaves were collected from plants grown under different treatments for 14 days. The leaves were thoroughly cleaned with de-ionized water to remove surface-adhered electrolyte. The samples were placed in a closed, glass tube containing 10 ml of de-ionized water. After a 24-h incubation at 25°C on a rotary shaker, electrical conductivity (L_t) was measured (Hanna Instruments, Canada). The samples were autoclaved at 120°C for 20 min and cooled to room temperature to determine the final electrical conductivity (L₀). Electrolyte leakage was calculated according to Lutts et al. (1996).

A membrane stability index (MSI) was determined according to the protocol published by Yadav et al. (2012). In this method the youngest, fully expanded leaves were collected from the plants grown under different treatments for 14 days and placed into 10 ml of de-ionized water in two sets, as triplicates. The electrical conductivity (C₁) of one set of samples was measured after heating at 40°C for 30 min in a water bath. The second set was boiled at 100°C for 10 min; electrical conductivity (C₂) was measured after cooling to room temperature. MSI was calculated using the following formula:

Lipid peroxidation was estimated by determining the concentration of malondialdehyde (MDA) in the leaves of the plants grown under different treatments for 14 days, after Hodges et al. (1999). Leaf material (0.5 g) was homogenized in 15 ml of 80% ethanol (EtOH), followed by centrifugation at 5,000 rpm at 4°C for 10 min. The supernatant (100 μl) and distilled water (900 μl) were mixed in a test tube with 1 ml of either (i) –TBA (thiobarbituric acid) solution including 20% (w/v) trichloroacetic acid (TCA) and 0.01% (w/v) butylated hydroxytoluene (BHT) or (ii) +TBA solution containing 0.65% (w/v) TBA with 20% (w/v) TCA and 0.01% (w/v) BHT. The mixture was vortexed, heated at 95°C in a dry bath for 25 min, cooled, and centrifuged at 5,000 rpm for 10 min. Absorbance was measured at 440, 532, and 600 nm. MDA equivalents were calculated using the following formula:

(1) [(Abs_532+TBA) – (Abs_600+TBA) – (Abs_532−TBA – Abs_600−TBA)] = A,

(2) [(Abs_440+TBA – Abs_600+TBA) 0.0571] = B,

(3) MDA equivalents (nmol/ml) = 10⁶ [(A – B) / 157,000].

In vivo localization and quantification of O2- and H₂O₂ were performed by histochemical staining with nitro-blue tetrazolium (NBT) and 3,3′-diaminobenzidine (DAB), as described by Yadav et al. (2012). To stain O2- in the leaves of the Z. mays grown under treatments C, T₁, T₂, and T₃, leaf samples were immersed in 1 mg ml⁻¹ of NBT in 10 mM phosphate buffer. After a 12-h incubation at room temperature, blue colored spots were observed, indicative of O2- formation. Similarly, H₂O₂ was stained by incubating leaf samples in 1 mg ml⁻¹ of DAB for 24 h. After incubation, brown colored spots were formed as a result of the reaction between DAB and H₂O₂.

O2- content was estimated in leaves of Z. mays, as described by Liu and Pang (2010). Leaf tissue was homogenized in 10 ml of 65 mM potassium phosphate buffer (pH 7.8) and centrifuged at 5,000 rpm for 10 min. The reaction mixture consisted of 0.9 ml of 65 mM phosphate buffer (pH 7.8) and 0.1 ml of 10 mM hydroxylamine hydrochloride, and 1 ml of the extract was incubated at 25°C for 20 min. Then, 17 mM sulfanilamide and 7 mM α-naphthylamine were added to the extract, the mixture was further incubated at 25°C for 20 min, and absorbance was read at 530 nm using a UV–VIS spectrophotometer. A standard curve (10–200 nmol) was prepared with NaNO₂ to calculate the production rate of O2-. The H₂O₂ content in leaf samples was measured as described by Yadav et al. (2012). Leaf tissue was extracted with cold acetone. Two milliliters of the extract was mixed with 0.5 ml of 0.1% titanium dioxide in 20% (v/v) H₂SO₄, and the mixture was centrifuged at 6,000 rpm for 15 min. The intensity of yellow color of the supernatant was measured at 415 nm using a UV–VIS spectrophotometer.

The effect of ANE treatments on the total soluble sugars and amino acids of Z. mays was determined according to the protocol described by Irigoyen et al. (1992) and Shukla et al. (2012), respectively. Leaf samples (100 mg) from all the treatments were frozen in liquid nitrogen and homogenized with mortar and pestle. The fine frozen powder was immediately suspended in 5 ml of 80% EtOH. The mixture was centrifuged at 5,000 rpm for 10 min at 4°C, and the pellet was re-extracted. Fractions were combined, and the final volume was made up to 15 ml. The total soluble sugars were analyzed by reacting 100 μl of alcoholic extract with 3 ml of freshly prepared anthrone reagent (150 mg anthrone in 100 ml of 72% (v/v) H₂SO₄). The mixture was placed in a boiling water bath for 10 min. After the mixture was cooled to room temperature, absorbance at 620 nm was measured. The content of total amino acids was determined by treating 1 ml of alcoholic extract with 1 ml of 0.2 M citrate buffer (pH 5.0), 1 ml of 80% EtOH, and 1 ml of ninhydrin (1%), followed by incubation at 95°C for 15 min. The samples were cooled, and the absorbance at 570 nm was measured.

For the quantification of secondary metabolites, Z. mays leaves were harvested from different treatments after 14 days. The samples were frozen in liquid nitrogen and homogenized with mortar and pestle. The fine frozen powder was immediately suspended in 70% methanol and centrifuged at 10,000 rpm for 10 min at 4°C. The total phenolics content was determined using Folin-Ciocalteu assay as described by Hodges and Lester (2006). One milliliter of extract was treated with 0.2 N Folin-Ciocalteu phenol reagent and vortexed. After incubation of the mixture for 5 min at room temperature, 1 ml of 7% Na₂CO₃ solution was added to the mixture. The mixture was incubated for 90 min at room temperature. After incubation, the absorbance against the reagent blank was determined at 550 nm. Total phenolics content was expressed as gallic acid equivalents (mg/g FW).

Total flavonoids content was determined according to Hichem et al. (2009). Briefly, 1 ml of plant extract was added to 4 ml of distilled water. The diluted samples were treated with 0.3 ml of 5% sodium nitrite (NaNO₂). After 5 min of incubation at room temperature, 0.3 ml of 10% aluminum chloride was added. Then, after 5 more minutes, 2 ml of 1 M sodium hydroxide (NaOH) was added to the mixture, and the final volume was made up to 10 ml. The solution was vortexed, and absorbance was measured against the reagent blank at 510 nm. The total flavonoids content was expressed as mg quercetin equivalents (QE).

To elucidate the role of ANE treatments in improving plant growth under P-limited conditions, real-time expression of genes involved in P homeostasis, carbohydrate metabolism, lipid metabolism, and secondary metabolism were performed. For gene expression analysis, experiments were conducted in a hydroponic system. Seven-day-old seedlings were inserted into a polystyrene disc floated on 1/2 MS (C), 1/2 MS + 0.01% ANE (T₁), 1/2 MS-P + 5 μM H₃PO₄ (T₂), and 1/2 MS-P + 0.01% ANE (T₃) in 300 ml Phyta jars. Leaf and root samples were harvested after 2 and 7 days of treatment. Total RNA was extracted using the RNAeasy kit (Qiagen, Germany) following the manufacturer's protocol. The quantity and purity of the total RNA were analyzed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). An amount of 2.5 μg RNA from each sample was treated with DNase I (Promega, USA), and then the synthesis of cDNA was performed using the Revert Aid cDNA reverse transcription kit (Thermo Scientific, USA). Real-time quantitative PCR (qPCR) was performed using the StepOnePlus Real-Time PCR system (Applied Biosystems). Actin and β-tubulin were used as reference genes. A list of the different primers used in this study is presented in Supplementary Table 1. The specificity of PCR amplification was checked at the end of the PCR cycles by melt-curve analysis. Each biological sample had three technical replicates, and the relative-fold expression was determined using the Livak (2^−ΔΔCt) method (Livak and Schmittgen, 2001).

Each experiment was carried out in triplicate, each experimental unit had six plants, and for each response variable, the average of the 18 values was used for statistical analysis. The results were presented as mean ± standard error. Data were analyzed using ANOVA, with a p ≤ 0.01 using the “Proc. mixed procedure,” of the SAS Institute, Inc. software version 9.3 (SAS Institute, Inc., Cary, NC, USA). When significant effects of treatments were found, multiple means comparison was carried out using Tukey's analysis with a 95% confidence interval. The significantly different mean values were represented by different alphabets.

Under low P conditions, the shoot and root growth of the Z. mays were significantly reduced, as compared with the control (Figure 1). The addition of ANE in the growth media improved the growth of the plant under P-limited conditions (Figure 1). Leaf area and total root length of the ANE-supplemented plants were significantly higher (p ≤ 0.01) in P-limited conditions than those of the control (Figures 1C,D). ANE-supplemented Z. mays plants grown under P-limited conditions showed higher fresh and DWs of shoots and roots, but not their percentage water content (PWC), than the control (Figure 2). The dry biomass of the shoot and root of ANE-supplemented Z. mays grown under P-limited conditions was found to be 4.33 and 1.29 times, respectively, higher than the plants grown in P-limited conditions alone (Figures 2B,E).

Mineral analysis was carried out on the leaves of the Z. mays plants grown under different treatments [1/2 MS (control, C), 1/2 MS + 0.01% ANE (T₁), 1/2 MS-P (T₂), and 1/2 MS-P + 0.01% ANE (T₃)] (Table 1). No significant differences were observed in the nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), zinc (Zn), and copper (Cu) contents of those plants grown in 1/2 MS and 1/2 MS + 0.01% ANE. P-limited conditions did not appear to have any influence on the K, Ca, and Na contents of the Z. mays. However, P-limited conditions significantly (p ≤ 0.01) reduced the N content in the plants, whereas ANE supplementation in the P-limited media increased N content by 18.3% in those plants. The application of ANE in the P-limited media significantly increased the P content, suggesting that ANE restored growth of those plants, in P-limited conditions, by regulating P homeostasis. Those plants grown in the presence of 1/2 MS-P had a reduction in Mg content of 30.2% and Zn by 26.25%, as compared with the control, whereas ANE treatments showed significantly less reduction in Mg content by 11.9% and Zn content by 15.3% under P-limited conditions, as compared with the control (Table 1).

P-limited conditions increased the Fe content by 101.6%, as compared with the control, but the increment of Fe content was less (i.e., 74.71%, as compared with the control) in those plants grown in the ANE-supplemented P-limited media (Table 1). The application of ANE significantly increased the uptake of B by 40% in those plants grown under normal conditions. P-limited conditions increased the uptake of B in plants grown in 1/2 MS-P (T₂) and 1/2 MS-P + 0.01% ANE (T₃), but the addition of ANE in P-limited media significantly increased the B content by 8.0%, as compared with the plants grown in P-limited conditions. ANE treatment increased the uptake of Mn by 7.6 and 11.5% in plants grown under the normal and P-limited conditions, respectively (Table 1).

ANE treatment showed no significant (p ≤ 0.01) effects on the chlorophyll a and b, carotenoid, and anthocyanin contents of leaves of Z. mays grown under normal conditions, as compared with the control (Figures 3A,B). The P-limited condition reduced the leaf content of chlorophyll a and b, whereas supplementation with ANE significantly increased the content of chlorophyll a and b in P-limited conditions. Carotenoid was also found to be higher in the ANE-treated plants grown in the P-limited media than in the plants grown in P-limited media alone (Figure 3C). The anthocyanin content was found to be increased by 173.1% as a result of the imposed P-limited conditions, whereas ANE-treated plants showed significantly less (98.3%) anthocyanin accumulation in plants grown under P-limited conditions (Figure 3D).

Z. mays plants treated with ANE showed a significant reduction of electrolyte leakage of leaves of those plants grown under P-limitation (Figure 4A). ANE reduced the electrolyte leakage by 35.3% in the leaves of Z. mays grown under P-limited conditions, as compared with the plants grown under P-limited conditions alone. MSI analysis revealed that the cell membranes of plants grown under P-limited conditions were more stable in those plants supplemented with ANE (Figure 4B). Membrane stability of the plants grown in 1/2 MS-P + 0.01% ANE was 68.2% higher than that of the plants grown in 1/2 MS-P (Figure 4B). To determine the impact of ANE treatment on the membrane lipids during P-limited conditions, the MDA content was measured in leaves of Z. mays grown under different treatments. No significant differences were observed in the MDA content of plants grown in 1/2 MS and 1/2 MS + 0.01% ANE. Compared with those plants grown in 1/2 MS-P, the MDA content was significantly reduced by 57.6% in ANE-supplemented plants, suggesting that lipid peroxidation of membrane lipids was significantly less than those grown in P-limited conditions (Figure 4C).

The impact of ANE treatments on oxidative damage induced by P-limited conditions was assessed by staining O2- and H₂O₂ in the tissues of leaves in treated Z. mays plants (Figure 5). Control and ANE-treatments showed similar patterns of staining, whereas P-limited conditions showed more staining of O2- and H₂O₂ than the ANE-treated plants (Figures 5A,B). These results suggested that ANE treatments helped plants to reduce the P-limited-induced accumulation of O2- and H₂O₂ in situ. These results were further confirmed by quantification of the tissue O2- and H₂O₂ contents (Figures 5C,D) that were significantly reduced in those ANE-treated plants grown under P-limited conditions, as compared with the control (Figures 5C,D).

P-starvation is known to influence the carbohydrate metabolism in plants (Karthikeyan et al., 2007). In maize plants grown on P-sufficient media, the level of total soluble sugars remained almost the same in both the control and ANE-supplemented plants. Plants grown on a P-limited medium showed a significant reduction in total soluble sugars content (Figure 6A). The supplementation of media with 0.01% ANE in P-limited media significantly increased the total soluble sugars content by 74%, as compared with the plants grown in P-starved medium (Figure 6A). The increased levels of total amino acids content were observed in those plants grown in 1/2 MS + 0.01% ANE, as compared with the control (Figure 6B). P-limitation showed a significant (p ≤ 0.01) reduction in amino acid metabolites. ANE application improved total amino acids content in those plants grown in P-limited media (Figure 6B). Imposition of a P-limited condition induced the total phenolics content, whereas the addition of ANE in the P-limited media reduced the total phenolics content in the plant (Figure 6C), suggesting that ANE treatment relieved plants from the deleterious effects of P-limitation. Similarly, the total flavonoids content was also significantly reduced in plants grown in ANE-supplemented P-limited media (Figure 6D). These results suggest that ANE supplementation in P-starved media improved plant growth by modulating the biochemical status of the plants.

To further elucidate the role of ANE treatments in ameliorating plant growth in P-impoverished conditions, the expression of genes involved in P homeostasis was evaluated in both the shoots and roots of Z. mays plants after 2 and 8 days of P-limited conditions. PHR1, a GARP-type MYB transcription factor, was significantly induced in the shoots and roots of the control, as well as ANE-supplemented plants grown under P-limited conditions, at both time-points investigated (Figure 7A). The induction of PHR1 in the roots and shoots of ANE-supplemented plants was significantly less (p ≤ 0.01) than that of the control. No significant change in the expression of PHT1 was observed in the shoots of Z. mays grown in the different treatments (Figure 7B). After 2 and 8 days, PHT1 was significantly induced in the roots of plants grown in 1/2 MS-P and 1/2 MS-P + ANE. The addition of ANE in P-limited media limited the transient up-regulation of PHT1, as observed in those plants grown under P-limitation alone (Figure 7B). PTF1, a P-starvation-induced, basic helix–loop–helix (bHLH) transcription factor, was induced in shoots after 2 days of imposition of P-limited conditions in treatments supplemented with ANE vs. control (Figure 7C). After 8 days, ANE treatments showed a reduction in the expression of PTF1 (Figure 7C). In roots, P-limited conditions induced the expression of PTF1 at both time-points, whereas ANE supplementation in P-starved media showed reductions in the expression of PTF1 (Figure 7C). These results suggested that experimental supplementation with ANE reduced the physiological severity of P-limitation.

SPX (SYG1/Pho81/XPR1) genes are known to play a key role in maintaining P homeostasis in plants (Secco et al., 2012). The expression of SPX1 was significantly induced in ANE-supplemented plants, as compared with the controls. From day 2 to day 8 of P-limitation, the expression of SPX1 was reduced from 5.94 times to 3.44 times in the shoots of Z. mays (Figure 7D). However, no change was observed in the expression of SPX1 in ANE-supplemented plants across the time-points. After 2 and 8 days of application of ANE, the expression SPX1 was increased by 1.46 and 2.26 times in the roots (Figure 7D). Under P-limited conditions, the SPX1 transcript was strongly expressed (5.04 times) at day 2, which was further increased to 6.18 times after 8 days of treatment. However, the addition of ANE in the 1/2 MS-P highly induced the expression of SPX1 in the roots at both time-points (Figure 7D). The expression of SPX3 remained unaltered in the shoots of plants grown in different treatments at both time-points. Similarly, plants grown in 1/2 MS (C) and 1/2 MS + ANE (T₁) did not show any change in the expression of SPX3 in their roots, across different time-points. P-limitation consistently increased the expression of SPX3 in roots, at both time-points. Overall, under P-limitation, the application of ANE resulted in strong induction (14.5 times) across the time-points, whereas P-limited conditions alone showed only 2.4 times induction from days 2 to 8 after treatment (Figure 7E).

P-starvation had a direct impact on photosynthesis and consequently reduced the carbon assimilation in those treatments. To cope with this situation, plants maneuver the expression of different genes involved in carbohydrate metabolism (Hermans et al., 2006). In shoots, under control conditions, ANE applications resulted in a significant induction (11.5 times) in the expression of SUC2, a gene involved in organ-specific sucrose transportation. After 2 days of imposition of P-limited conditions, the expression of SUC2 induced the expression by 8.5 times in the shoots, as compared with the control, whereas supplementation of ANE in the P-limited media further induced the expression by 17.2 times. However, after 8 days, the expression pattern of SUC2 was found to be 3.18 times higher in the shoots of ANE-supplemented plants grown in P-limited media than in those plants grown in P-limited media alone (Figure 8A). In the roots, initially, after 2 days, SUC2 was down-regulated in all treatments, as compared with the control, whereas at the later stage, all plants showed induction of SUC2 in all treatments, as compared with the control (Figure 8A). The Glucose 6P/P translocator (G6Ptr) involved in the conversion of Glucose 6P to starch was differentially regulated in the shoots and roots of Z. mays grown under P-limited conditions (Figure 8B). After 2 days of P-limitation, ANE supplementation induced the expression of G6Ptr in the shoots (Figure 8B), whereas after 8 days, no differences in expression patterns were observed in any of the treatments. In roots, the transcript accumulation of G6Ptr was found to be higher at both time-points in those Z. mays plants that were treated with ANE and grown under P-limited conditions than the control (Figure 8B). Initially, no changes in the expression patterns of sucrose phosphate synthase (SPS) were observed in the shoots and roots of ANE-treated, P-limited plants. However, after 8 days of P-limitation, ANE-supplemented plants showed a significant induction in the expression of SPS, as compared with the control (Figure 8C). ANE applications significantly stimulated the expression of pyruvate kinase 1 (PK1) in the roots and shoots of plants grown under P-limited conditions, at both time-points, as compared with the plants grown in P-limited media alone (Figure 8D). ADP-glucose pyrophosphorylase (AGPase) was significantly induced in the shoots of ANE-treated, P-limited plants at both time-points. In the roots, after 2 days of P-limitation, the expression of AGPase was 2.6 times higher in the ANE treatments, which declined to 1.33 times after 8 days, than in the control (Figure 8E). Likewise, phosphoenolpyruvate carboxylase (PEPCase), an important gene involved in carbon assimilation, was found to be significantly up-regulated in the shoots and roots of ANE-supplemented, P-limited plants, at both time-points (Figure 8F). These results suggest that the application of ANE regulated the carbon assimilation and better plant growth resulted under P-limited conditions.

To survive in P limiting conditions, plants go through significant alterations in their membrane lipid compositions by regulating the expression of different genes involved in lipid metabolism. Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main components of glycolipids of the thylakoid membrane. MGDG synthase (MGDGS) and DGDG synthase (DGDGS) catalyze the formation of MGDG and DGDG. P-limited conditions induced the expression of MGDGS and DGDGS in the shoots and roots of Z. mays at both time-points (Figures 9A,B). ANE supplementation increased the expression of MGDGS in the shoots of Z. mays grown under P-limited conditions, by 2.6 and 1.58 times at 2 and 8 days, respectively. Similarly, in the roots, the expression of MGDGS was significantly induced at both time-points in ANE-treated, P-limited plants (Figure 9A). After 2 days of P-limited conditions, no changes in the expression pattern of DGDGS were observed in either the shoots or roots of the treatments (Figure 9B). Prolonged exposure of P-limited conditions in the presence of ANE increased the transcript accumulation of DGDGS in shoots, whereas no substantial differences were observed in the roots, as compared with the control (Figure 9B). Phosphatidylinositol (PIS) is a key enzyme involved in phospholipid pathways. After 2 days, the expression of PIS was significantly reduced in plants grown in 1/2 MS-P (T₂) and 1/2 MS-P + ANE (T₃), as compared with those plants grown in 1/2 MS (C) and 1/2 MS + ANE (T₁) (Figure 9C). ANE significantly induced the expression of PIS after 8 days, as compared with the control plants (Figure 9C). P-limited conditions, however, increased the expression of PIS from 0.7 to 2.5 times from day 2 to 8, whereas the treatment of ANE application in P-starved media increased expression from 0.6 to 4.2 times from day 2 to 8 (Figure 9C). Notably, in the roots, no difference was observed in the expression of PIS among all the treatments at both time-points (Figure 9C). In the shoots of those plants grown under P-limited conditions, the expression of fatty acid desaturase 7 (FAD7) increased from 2.2 to 5.1 times between time-points. This trend was not observed in plants grown under P-limited conditions with ANE treatment (Figure 9D). In roots, FAD7 was also induced at both the time-points by P-limited conditions. By contrast, the expression was reduced from 6 to 2.6 times from day 2 to 8 (Figure 9D). In the roots of ANE-treated plants, under P-limited condition, FAD7 was also induced at both time-points, but to a lesser degree than those plants grown in P-limited conditions (Figure 9D). The expression of diacylglycerol kinase 1 (DGK1) was significantly induced (2.2 times) in the shoots of plants treated with ANE, as compared with the control conditions (Figure 9E). P-limited conditions increased the expression of DGK1 in the shoots of plants grown with ANE, as well as without ANE, but the expression was 2.81 times higher in ANE-supplemented plants grown under P-limited conditions than in the plants grown under P-limited conditions alone (Figure 9E). In roots, DGK1 was significantly induced at both time-points by P-limited conditions; however, ANE supplementation in P-starved media induced DGK1 by 2.25 times and 1.4 times at 2 and 8 days, respectively (Figure 9E). These results suggest that ANE supplementation regulated the expression of genes involved in lipid metabolism that helped treated plants to grow better under P-limited conditions.

After 2 days of treatment, Bronze 2 (Bz2), an anthocyanin biosynthetic gene in maize, was significantly down-regulated (2 times) in the shoots of plants grown in the 1/2 MS media supplemented with ANE, as compared with the control plants (Figure 10A). Bz2 was induced by 1.86 times in plants grown for 2 days in 1/2 MS-P (T₂), as compared with the plants grown in 1/2 MS-P + ANE (T₃) (Figure 10A). The expression of Bz2 was reduced in the shoots after 8 days under P-limited conditions. However, no changes were observed in shoots of the ANE-treated plants under P-limited conditions (Figure 10A). In roots, after 2 days of treatment, Bz2 was down-regulated in all treatments, as compared with the control, whereas maximum reduction in expression was observed in those plants grown in 1/2 MS + ANE (T₁) (Figure 10A). Prolonged exposure of P-limited conditions increased the expression of Bz2 in those plant roots. Addition of ANE in P-limited media significantly increased the expression of Bz2 in the roots by 2.7 times, as compared with the plants grown under P-limited conditions alone (Figure 10A). Flavonol synthase 1 (FLS1), an important gene involved in flavonol biosynthesis, was differentially regulated in the shoots and roots of Z. mays in all treatments (Figure 10B). Initially, no change in the expression of FLS1 was observed (Figure 10B). However, after 8 days under P-limited conditions, ANE-supplemented plants showed 1.8 times increased expression of FLS1, as compared with the P-limited plants alone (Figure 10B). No significant changes were observed in the expression of FLS1 in the roots of plants grown under different treatments, at both time-points (Figure 10B). The expression of cinnamyl alcohol dehydrogenase (ZmCAD) remained unchanged in the shoots and roots of Z. mays in all treatments after 2 days (Figure 10C). Expression analysis revealed that the transcript accumulation of CAD increased in the shoots of ANE-supplemented plants grown for 8 days under P-limited conditions. In roots at 8 days after treatment, expression was significantly reduced in the ANE-treated plants grown under P-limited conditions (Figure 10C). Under P-limited conditions, ANE supplementation significantly increased the expression of dihydroflavonol-4-reductase (ZmDHFR) and chalcone synthase (ZmCHS) in the shoots of Z. mays at both time-points (Figures 10D,E). These results allow us to draw a conclusion that ANE regulates secondary metabolism of plants grown under P-limited conditions and help the plant to reduce the deleterious effect of P-limitation.