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. 2020 Dec 12;26(12):2503–2520. doi: 10.1007/s12298-020-00914-y

Foliar spray of Auxin/IAA modulates photosynthesis, elemental composition, ROS localization and antioxidant machinery to promote growth of Brassica juncea

Anayat Rasool Mir 1, Husna Siddiqui 1, Pravej Alam 2, Shamsul Hayat 1,✉
PMCID: PMC7772134  PMID: 33424161

Abstract

Auxins (Aux) are primary growth regulators that regulate almost every aspect of growth and development in plants. It plays a vital role in various plant processes besides controlling the key aspects of cell division, cell expansion, and cell differentiation. Considering the significance of Aux, and its potential applications, a study was conducted to observe the impact of indole acetic acid (IAA), a most active and abundant form of Aux on Brassica juncea plants growing under natural environmental conditions. Different concentrations (0, 10−10, 10−8, 10−6 M) of IAA were applied once in a day at 25-day stage of growth for 5 days, consecutively. Various parameters (growth, photosynthetic, biochemical, oxidative biomarkers and nutrient composition) were assessed at different days after sowing (DAS). Scanning electron microscopy (SEM) of leaf stomata, reactive oxygen species (ROS) localization in leaf and roots, and confocal microscopy were also conducted. The results revealed that all the IAA concentrations were effective in growth promotion and ROS reduction, however, the 10−8 M of IAA exhibited the maximum improvement in all the above mentioned parameters as compared to the control.

Keywords: Confocal microscopy, Histochemical studies, Photosynthesis, Stomatal movement, SEM–EDX

Introduction

Auxins (Aux) are the primary plant growth regulator that functions virtually in every perspective of plant growth and development (Davies 2004; Weijers and Wagner 2016). Their existence as a growth regulator was first demonstrated by Charles and Darwin (Darwin and Darwin 1880), and later on, was isolated by Fritz Went (1935). Aux are indolic compounds that are structurally related and synthesized from amino acid tryptophan (Paque and Weijers 2016). Rigorous research on Aux in past decades have made remarkable advancement in the elucidation of its transcriptional pathway (Weijers and Wagner 2016), mechanisms involved in uni-directional transport (Adamowski and Friml 2015), and its bio-synthesis (Korasick et al. 2013). In nutshell, it can be said that various genes are regulating the Aux biosynthesis, catabolism, conjugation, and conjugate hydrolysis (Normanly 2010; Zhao 2012; Korasick et al. 2013; Ljung 2013). It regulates various other phytohormones (Vert and Chory 2011) and also performs a vital role in plant interactions with various beneficial microorganisms (Boivin et al. 2016). Aux regulates the gene expression and cellular responses through Aux/IAA proteins together with Aux response factors (ARFs), to induce the growth and development in plants (Guilfoyle et al. 1998; Ulmasov et al. 1999; Tiwari et al. 2003). Recent investigations have demonstrated that Aux responses occur through repression of Aux/IAA repressors and ubiquitin–proteasomic degradation of Aux receptors by increased concentration of Aux in cells (Benfey 2002; Hellmann and Estelle 2002). The degeneration of Aux/IAA repressors enables the ARF activators to repress/stimulate the expression of various Aux-responsive genes (Benfey 2002). IAA acts as a key substance in the regulation of plant growth, and various other related physiological functions and shows a positive relation with these-growth attributes (McKay et al. 1994). It regulates cell division, cell elongation, and development of adventitious root, embryogenesis, and leads to cell wall softening at lower concentration (Taiz and Zeiger 2006). IAA increases pigment content, photosynthetic rate, stomatal conductance, and accumulation of sugars such as glucose, fructose, and total soluble sugars in plants (Hayat et al. 2009; Singh and Prasad 2015; Li et al 2019). In addition, Aux enhances photosynthetic rate by increasing the density of leaf veins coordinated through their well-organized placement which drives the improvement in leaf photosynthetic capacity (McAdam et al. 2017). IAA application also enhances nitrate reductase activity and favors the growth as reported in Solanum melongena (Hayat et al. 2006). IAA as well as its precursors such as L-tryptophan (Trp) and indole (Ind) increases the contents of mineral nutrients in roots and leaves of plants (San-Francisco et al. 2005; López et al. 2007). Moreover, Aux enhances enzymatic (ascorbate peroxidase, superoxide dismutase, catalase) and non-enzymatic (glutathione, ascorbate) antioxidants, and suppresses various ROS levels including lipid peroxidation and hydrogen peroxide (Pasternak et al. 2005; Piotrowska-Niczyporuk and Bajguz 2014).

Brassica was selected for research because of its agricultural significance and the extensive amount of information available regarding its physiology and biochemistry. It is one of the major oil yielding crops in India and ranks third in production and area coverage after China and Canada. The yield is very less in contrast to other countries cultivating rapeseed-mustard. Considering the growth promoting role of Aux in plants, this research was designed to examine the role of different Aux concentrations on growth, physiological, photosynthetic, and various biochemical aspects in Brassica juncea. In addition, its impact on nutrient metabolism and detoxifying property against reactive oxygen species (ROS) has also been evaluated.

Materials and methods

Hormone preparation

The desired amount of IAA was dissolved to make the stock solution and adjusted with DDW to get the final volume of 100 ml. The stock was diluted to get the required concentrations used in the experiment.

Biological material

The seeds of Brassica juncea var. Varuna were obtained from Aligarh Seed Store, Aligarh, U.P., India and uniform sized seeds were selected for the experiment.

Experimental design and treatments

A randomized block design was adopted with 20 earthen pots stuffed with soil and farmyard manure maintained in the ratio of 6:1. The surface sterilized seeds were sown carefully in the last week of October, and experiment was performed in the net house of the Department. Aux (10−10, 10−8, and 10−6 M) treatments were applied as a foliar spray at 25 days after sowing (DAS), consecutive for 5 days. Control plants were treated with DDW. Four rows were arranged containing five replicates of all treatment and every replicate holds 3 plants per pot. The plants were sampled at various intervals (30, 45, or 60 days) to determine the several traits discussed below.

Growth characteristics

The selected plants were taken out smoothly from pots and washed in a water-filled bucket to eradicate the adherent soil on the roots. By using a metric scale, the growth parameters like length of root and shoot length were measured and their fresh and dry mass was determined by electronic balance (Varbal 100 super, Varanasi, Balance works, Varanasi, India). Leaf area was measured using leaf area meter (AM 350, ADC Bio Scientific Ltd. Hoddesdon, Herts, UK) and was expressed in cm2.

Relative water content (RWC)

The status of water in plant was estimated by determining the RWC in the leaf. Entirely extended leaf was taken off from all treatment between 11:00 AM and 12:00 noon following the procedure demonstrated by Hayat et al. (2007). Circular leaf discs of 2 cm diameter were prepared and their fresh weight was immediately measured and dipped in a petri dish filled with DDW for 24 h. The petri dishes were enclosed in a shaded sheet to maintain absolute darkness. After 24 h, the leaf samples were plucked and weighed to determine their turgor mass. The leaf discs were further dried at 80 °C for 48 h to determine their dry mass. RWC was calculated by the following formula:

RWC%=FW-DWTW-DW∗100

where FW is fresh weight, DW is dry weight and TW is turgid weight.

SPAD chlorophyll

The content of chlorophyll in the leaves was determined using SPAD chlorophyll meter (SPAD-502; Konica, Minolta sensing, Inc., Japan).

Scanning electron microscopy (SEM)

A method reported by Siddiqui et al. (2018) was followed to perform SEM imaging of leaf stomata. Fresh leaf samples were taken and immediately anchored with 0.1 M sodium cacodylate buffer (pH 7.3), 2% paraformaldehyde, and 2.5% glutaraldehyde for 2 h. The samples were dehydrated with graded concentration of ethanol (50%, 70%, 80%, 90%, and 100%). Fully dehydrated leaves were glazed with gold–palladium in a sputter-coated instrument (JOEL JFC-1600, Tokyo, Japan) and stomatal aperture images were captured using scanning electron microscopy (JEOL JSM-6510, Tokyo, Japan).

Photosynthetic characteristics

Net photosynthetic rate (PN), stomatal conductance (gs), transpiration rate (E), and internal carbon dioxide concentration (Ci) were analyzed using infrared gas analyzer (IRGA) portable photosynthetic system (LI-COR 6400, LI-COR, Lincoln, NE, USA). The IRGA was calibrated to maintain the air temperature at 25 °C, relative humidity was adjusted to 85%, CO2 concentration at 600 μmol mol−1, and PPFD was maintained at 800 μmol mol−2 s−1, respectively.

Nitrate reductase (NR) activity

The activity of NR in leaves was assessed using the method of Jaworski (1971). Freshly collected leaves were sliced into pieces and soaked in the reaction mixture containing KNO3, isopropanol, and phosphate buffer adjusted at pH 7.5, at 30 °C for 2 h. Afterward, a solution of sulfanilamide and N-1-naphthylethylene diamine hydrochloride solutions were added carefully. The optical density was measured on a spectrophotometer at 540 nm. The activity of NR was computed on a fresh mass basis as n mole NO2 g−1 FM s−1.

Carbonic anhydrase (CA) activity

The method of Dwivedi and Randhawa (1974) was followed to determine the activity of CA. Fresh leaf sample was made into small strips and immersed into a reaction mixture containing 0.2 M cysteine hydrochloride solution. The solution is filtered, and to this filtrate, phosphate buffer (pH 6.8), 0.2 M NaHCO3, and bromothymol blue are added. Methyl red was used as an indicator, and the solution was titrated against 0.05 N HCL. The result was expressed as mol CO2 kg−1 (FM) s−1.

Leaf proline content

Proline content was estimated in fresh leaf by the method described by Bates et al. (1973). Freshly collected leaf sample was homogenized in 3% sulphosalicylic acid, followed by addition of an equal volume of glacial acetic acid and freshly prepared ninhydrin solutions. The sample was boiled at 100 °C for 2 h and consequently cooled down. 5 ml of toluene layer was added to each tube. The absorbance of immiscible toluene layer was measured spectrophotometrically at 520 nm.

Total soluble sugars

Total soluble sugar in the leaf was determined by the method of Dubois et al. (1956). Leaf powder (50 mg) was dissolved in 80% ethanol and centrifuged at 10,000 rpm. Supernatant was collected, and absorbance was recorded spectrophotometrically at 485 nm. The content of sugar was calculated using a standard curve of D-glucose.

Reducing sugars

The method of Sumner (1935) was followed to determine the reducing sugar content in leaves. Leaf powder (50 mg) was homogenized in 80% ethanol and centrifuged at 10,000 rpm. The optical density of the supernatant was read at 560 nm. Reducing sugar content was estimated from the standard curve of D-glucose.

Protein analysis

Protein content was determined as described by Bradford (1976). The optical density was recorded at 595 nm by spectrophotometer.

Estimation of superoxide (O2−) level

Superoxide anion content was estimated according to our previous study (Siddiqui et al. 2018) and compared by the standard curve of sodium nitrite. The O2− anion content was expressed as µmole g−1 F.W.

Localization of O2−

The degree of generation of O2−was determined by histochemical staining method, using Nitro-blue tetrazolium (NBT) to stain the leaves (Kaur et al. 2016) with some modifications. Fresh leaf sample was dipped in NBT solution for 8 h at normal room temperature. This was followed by boiling the leaf samples in concentrated ethanol for 2 h to remove the chlorophyll pigments. The leaf sample was cooled down and dipped in 20% glycerol solution. Pictures were taken using stereomicroscope.

Determination of hydrogen peroxide (H2O2)

The hydrogen peroxide content was estimated as described earlier (Patterson et al. 1984). Freshly collected leaf (0.5 g) was extracted in acetone and centrifuged at 5000 × g for 15 min. The supernatant was collected and added with 20% titanium chloride prepared in conc. HCl to which Ammonia solution (17 M) was added. The precipitate was collected and repetitively rinsed with acetone followed by addition of 2 N H2SO4. Absorbance was measured at 410 nm. H2O2 content was calculated by preparing the standard curve using H2O2 and expressed as μmole g−1 F.W.

Localization of hydrogen peroxide

The localization of superoxide anions was visualized as earlier (Kaur et al. 2016) with some changes. Freshly collected leaves of equal size immersed in 3,3′- diaminobenzidine solution at acidic pH (3.8) for about 8 h at room temperature. The leaves were then taken out and boiled in concentrated ethanol to remove the chlorophyll pigments and was followed by transferring the leaves in 20% glycerol. Pictures were taken using stereomicroscope.

Lipid peroxidation

The method of Heath and Packer (1968) was followed for the estimation of Lipid peroxidation in terms of malondialdehyde (MDA) content. Fresh leaf sample was extracted in 0.1% trichloroacetic acid (TCA) and homogenized at 10000×g for 15 min. Supernatant was collected, followed by the addition of 20% TCA consisting of 0.5% thiobarbituric acid. The sample mixture was incubated at 95 °C for 30 min and subsequently cooled. The resulting reaction mixture was homogenized at 1000×g for 15 min at 4 °C. Optical density of the supernatant was measured at 532 nm.

Antioxidant enzymes activity

The method of Ramiro et al. (2006) was used for the estimation of antioxidant enzymes i.e., superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX). Freshly taken leaf sample (0.5 g) was crushed in 5 ml phosphate buffer (50 mM, pH 7.0) comprising of 1% polyvinylpyrolidone (PVP). The resulting mixture was homogenized at 12,000×g for 20 min. The extraction process was carried out at 4 °C.

Leaf SOD activity

Methodology of Kono (1978) was followed to determine the activity of SOD. A solution was prepared consisting of 50 mM buffer phosphate (pH 7.8), 130 mM methionine, 75 μM nitro blue tetrazolium (NBT), 0.1 mM ethylene diamine tetra acetic acid (EDTA), and 20 μM riboflavin. The resulting reaction mixture was set aside in light for 10 min, and the optical density was measured at 560 nm.

Leaf CAT activity

Methodology of Aebi (1984) was followed to estimate the activity of CAT. A reaction mixture was prepared consisting of 100 mM phosphate buffer (pH 7.8), 10 mM H2O2 and 100 μl of enzyme extract. The CAT activity was measured spectrophotometrically at 240 nm by disappearance of H2O2.

Leaf POX activity

The POX activity was estimated by the method of Chance and Maehly (1955) with slight changes. The 3 ml of reaction mixture was prepared comprising of pyrogallol phosphate buffer, 1% H2O2, and 1 ml of enzyme extract. POX enzyme activity was assessed by recording the absorption at 420 nm.

Elemental composition

Various element [carbon (C), magnesium (Mg), phosphorus (P), sulphur (S), potassium (K)and calcium (Ca)] was analyzed at 45 DAS using SEM–EDX technique according to our previous study (Siddiqui et al. 2018).

Confocal laser scanning microscopy

Cell viability was studied by immersing the roots in the propidium iodide solution. The roots were then taken off and washed with DDW. Glass slides were prepared and were viewed in the confocal laser scanning microscope (Zeiss, LSM 780, Tokyo, Japan). Reactive oxygen species (ROS) localization was visualized in roots by dipping the samples in DCF-DA dye. These root samples were taken off and mounted on glass slides. Prepared slides were viewed by confocal laser scanning microscope (Zeiss, LSM 780, Tokyo, Japan).

Statistical analysis

SPSS ver. 17 for Windows (IBM Corporation, New York, USA) was used for statistical analysis. Analysis of variance (ANOVA) and standard error was calculated using 5 replicates (n = 5) to access the least significance difference (LSD) between treatment means with the level of significance at p ≤ 0.05. Duncan’s multiple range test was used to separate the means.

Results

Growth characteristics

The foliar application of IAA significantly improved all the growth biomarkers (length, fresh and dry mass of root and shoot and leaf area; Fig. 1A–F) as compared to control irrespective of concentrations. 10−8 M of IAA proved most effective in elevating the growth attributes over all the other concentrations. The maximum increase in shoot length over the control was about 17.51%, 41.0%, and 36.95% and for root length was 8.51%, 18.97%, and 14.44%, respectively at 30, 45, and 60 DAS. The minimum improvement was observed in plants sprayed with 10−6 M of IAA where, the increase over control in shoot length was about 6.38%, 25.85%, and 18.91%, and for root length was 2.22%, 8.29%, and 5.44% at respective DAS as mentioned above. The highest improvement in fresh mass of shoot (32.29%, 56.15% and 49.23%) and dry mass (14.46%, 37.34%, and 33.38%) was reported in 10−8 M of IAA at 30, 45, and 60 DAS compared to control. Minimum increase in shoot fresh mass (9.54%, 23.96%, and 20.22%) and dry mass (6.0%, 17.28%, and 15.58%) was recorded in plants treated with 10−6 M of IAA over the control respectively. Root also displayed a similar pattern of increase in fresh mass (25.80%, 47.80%, and 32.43%) and dry mass (17.80%, 26.80%, and 23.36%) over the control plants. Similarly the minimum increase over the control was reported in 10−6 M of IAA treated plants. The increasing percentage over the control in root fresh mass was about 9.90%, 19.96%, and 17.38% and dry mass was about 6.85%, 11.34%, and 11.27% at 30, 45 and 60 DAS respectively. Additionally, the spray of IAA markedly increased the leaf area, as compared to the controls. The maximum leaf area was shown by plants treated with 10−8 M of IAA, which was about 4.9%, 23.85%, and 20.94% more whereas, a minimum increase was recorded in plants treated with 10−6 M IAA, which was about 2.10%, 10.39%, and 8.10% at 30, 45 and 60 DAS, respectively more than their respective controls (Fig. 2E).

Fig. 1.

Fig. 1

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Effect of different doses of IAA (0, 10−10, 10−8 or 10−6 M) on A shoot length, B root length, C shoot dry mass, D root fresh mass, E shoot fresh mass, F root dry mass of Brassica juncea at 30, 45 and 60 days stage of growth.Values of bars with different letter are significantly different, P < 0.05 Duncan’s multiple range test

Fig. 2.

Fig. 2

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Effect of different doses of IAA (0, 10−10, 10−8 or 10−6 M) on A peroxidase activity, B superoxide dismutase activity, C catalase activity, D SPAD, E Leaf area, and F RWC of Brassica juncea at 30, 45 and 60 days stage of growth. Values of bars with different letter are significantly different, P < 0.05 Duncan’s multiple range test

Relative water content

IAA application increased RWC at all the sprayed concentrations, over the DDW treated controls (Fig. 2F). Maximum response was observed at 10−8 M IAA treatment. The increase over control was about 15.16%, 28.97%, and 20.59% at 30, 45 and 60 DAS, respectively. The minimum increase compared to control was reported in plants sprayed with 10−6 M IAA. The improvement in RWC over the control was about 8.16%, 17.92% and 10.77% at 30, 45 and 60 DAS, respectively.

SPAD chlorophyll values

Aux significantly elevated the SPAD chlorophyll level in all the treatments compared to control, with 10−8 M IAA concentration showing the best stimulatory effect on chlorophyll content (Fig. 2D). The maximum increase over control was 13.27%, 18.90%, and 17.87% at 30, 45 and 60 DAS, respectively. The minimum increase compared to the control was reported in plants sprayed with 10−6 M IAA and was about 6.40%, 9.14% and 7.06% at 30, 45 and 60 DAS, respectively.

Gaseous exchange parameters

Figure 3A–D clearly indicates that PN, gs, E, and Ci were markedly improved by all the treatments sprayed over the control. The maximum improvement in these parameters was obtained in plants sprayed with 10−8 M of IAA, whereas minimum enhancement was reported in plants treated with 10−6 M IAA. The highest increase in PN was about (10.43%, 28.45% and 25.72%), E (26.60%, 37.05%, and 27.20%), Ci (18.40%, 25.24%, and 23.21%) and gs (39.22%, 50.0% and 42.46%) respectively at 30, 45 and 60 DAS. The minimum increase in PN was about (2.66%, 16.12% and 14.62%), E (8.01%, 11.42% and 10.26%), Ci (6.60%, 9.51% and 6.81%) and gs (25.49%, 37.50% and 24.66%) respectively at 30, 45 and 60 DAS over the control.

Fig. 3.

Fig. 3

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Effect of different doses of IAA (0, 10−10, 10−8 or 10−6 M) on A net photosynthetic rate, B stomatal conductance, C Internal CO2 concentration, D Transpiration rate and E Total soluble sugar, F reducing sugar content of Brassica junceaat 30, 45 and 60 days stage of growth.Values of bars with different letter are significantly different, P < 0.05 Duncan’s multiple range test

NR activity

All the tested concentrations significantly elevated the activity of NR as compared to the control plants (Fig. 4C). The maximum activity was obtained from the plants treated with 10−8 M of IAA. The improvement was about 17.12%, 21.88%, and 19.75% respectively at 30, 45 and 60 DAS, compared to the control. Minimum improvement in NR activity was determined in plants treated with 10−6 M IAA. The percentage increase was about 3.29%, 6.06%, and 4.98% compared to their respective controls at 30,45 and 60 DAS.

Fig. 4.

Fig. 4

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Effect of different doses of IAA (0, 10−10, 10−8 or10−6 M) on A Carbonic anhydrase, B proline, C Nitrate reductase, D MDA, and E Protein content, of Brassica junceaat 30, 45 and 60 days stage of growth.Values of bars with different letter are significantly different, P < 0.05 Duncan’s multiple range test

CA activity

The Fig. 4A clearly shows the enhanced activity of CA in IAA treated plants at 30, 45 and 60 DAS with respect to their control. The CA activity demonstrated a substantial increase of 17.12%, 21.88% and 19.75% over their respective controls at 30, 45 and 60 DAS in 10−8 M. The minimum increase in CA activity was recorded in 10–6 M which was about 3.24%, 6.44% and 5.24% contrary to the control respectively at 30, 45 and 60 DAS.

Total carbohydrate and reducing sugar content

It is evident from Fig. 3E and F that the total carbohydrate content and reducing sugar showed an enhancement as the growth progressed from 30 to 60 days. The results were further improved by the foliar application of IAA and highest increase was reported in 10−8 M of IAA, which was about 10.56%, 18.69%, and 17.72% and 8.56%, 19.08%, and 16.75% more for total carbohydrate and reducing sugar content at 30, 45 and 60 DAS, respectively. The minimum increase in total carbohydrate (4.72%, 9.81%, and 8.63%) and reducing sugar (5.42%, 11.27%, and 6.82%) content was observed in 10−6 M IAA treated plants respectively at 30, 45 and 60 DAS compared to the control.

Proline content

Foliar spray of IAA significantly elevated the accumulation of proline concentration in plants (Fig. 4B). The maximum proline accumulation was reported in plants supplied with 10−8 M IAA. The percent increase in 10−8 M IAA treated plants over their respective control plants was about 18.46%, 26.97%, and 23.22% at 30, 45 and 60 DAS respectively, however, the minimum increase was shown by plants sprayed with 10−6 M IAA, which was about 8.71%, 13.25%, and 9.53% over the control at 30, 45 and 60 DAS.

Protein content

Foliar spray of IAA significantly increased the accumulation of protein content in plants (Fig. 4E). The highest protein content was reported in plants treated with 10−8 M IAA. The percent increase in 10−8 M IAA treated plants over their respective controls was about 6.60%, 14.28%, and 12.95%, at 30, 45, and 60 DAS respectively. The minimum improvement was reported in plants sprayed with 10−6 M IAA, which was about 3.52%, 6.49%, and 3.24% over the control at respective DAS as mentioned above.

Antioxidant enzyme activities

IAA significantly enhanced the antioxidant enzyme activities at all IAA levels tested when compared to control (Fig. 2A–C). The activity of enzymatic antioxidants (i.e., SOD, CAT, and POX) displayed a substantial increase as the growth proceeded from 30 to 60 DAS. Their activity was increased further by the foliar application of IAA at all the concentrations. The maximum activity was shown by plants sprayed with 10−8 M IAA and the value increased for SOD by 23.33%, 30.23%, and 27.94%, CAT by 18.73%, 29.46%, and 24.47% and POX by 16.93%, 27.39%, and 25.82% compared to their corresponding control plants at 30, 45 and 60 DAS respectively. The minimum improvement in the activity of SOD (11.67%, 13.18%, and 11.76%), CAT (7.45%, 12.17%, and 8.03%) and POX (9.77%, 18.78%, and 15.92%) was reported in 10−6 M IAA treated plants at 30, 45, and 60 DAS respectively compared to their respective controls.

MDA content in leaves

At all the IAA tested concentrations the MDA content was significantly reduced as compared to the DDW treated control plants (Fig. 4D). The maximum MDA content was reported in control plants. The utmost decrease was obtained by the plants sprayed with 10−8 M IAA over the control which was about 8.93%, 22.35%, and 14.73%, whereas, minimum decline (4.22%, 8.83%, and 3.13%) was reported in plants treated with 10–6 M IAA, respectively at 30, 45 and 60 DAS.

Stomatal response

The exogenous application of IAA widened the stomatal aperture as compared to the control plants. The 10−8 M IAA (3.65 µm) concentration proved more efficient in broadening the stomatal aperture over the control (1.65 µm) at 45 DAS (Fig. 5A, B).

Fig. 5.

Fig. 5

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Scanning electron microscope (SEM) images of stomata: Response of stomatal aperture at 45 days old Brassica juncea leaves treated with IAA, A Represent control and B Represents 10−8 M IAA at 4000×magnification

Elemental composition

Elemental composition displayed a considerable improvement at 45 DAS in the presence of IAA in a dose dependent manner. The highest improvement was recorded at 10−8 M IAA concentration, wherein the increment for C (10.92%), Mg (23.08%), P (18.75%), S (21.43%), K (28.79%), and Ca (20.26%) content at 45 DAS over the control was observed (Table 1; Fig. 6). The minimum increase in element composition was observed in 10−6 M IAA treated plants. The increasing percentage for C (5.04%), Mg (7.69%), P (6.25%), S (9.52%), K (12.12%), and Ca (10.46%) content at 45 DAS over the control was recorded. Our findings were further visualized by mapping the elements (Fig. 6).

Table 1.

Effect of different doses of IAA (0, 10−10, 10−8 or10−6 M) on the percent dry weight of different elements i.e., carbon, magnesium, phosphorus, sulphur, potassium and calcium in Brassica juncea at 45-day stage of growth.

Treatments Carbon
(% dry weight)		 Magnesium
(% dry weight)		 Phosphorus
(% dry weight)		 Sulphur
(% dry weight)		 Potassium
(% dry weight)		 Calcium
(% dry weight)
Water sprayed (control) 36.89 ± 0.18d 0.13 ± 0.002d 0.42 ± 0.012d 0.48 ± 0.008d 0.66 ± 0.019d 1.53 ± 0.059d
IAA (10−6 M) 38.75 ± 0.27c 0.14 ± 0.003c 0.46 ± 0.013c 0.51 ± 0.012c 0.74 ± 0.013c 1.69 ± 0.090c
IAA (10−8 M) 40.92 ± 0.12a 0.16 ± 0.003a 0.51 ± 0.012a 0.57 ± 0.009a 0.85 ± 0.014a 1.84 ± 0.062a
IAA (10−10 M) 39.79 ± 0.16b 0.15 ± 0.003b 0.48 ± 0.010b 0.54 ± 0.008b 0.79 ± 0.009b 1.77 ± 0.057b
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Values of bars with different letter are significantly different, P < 0.05 Duncan’s multiple range test

Fig. 6.

Fig. 6

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SEM–EDX graphs along with mapping in various IAA (0, 10−10, 10−8 or 10−6 M) treated plants showing elemental analysis in dried leaves of Brassica junceaat 45 day stage of growth. Each element is mapped with a particular color

H2O2 content

It is clear from the Fig. 2C that leaf H2O2 content markedly decreased upon application of AUX. The highest H2O2 concentration was observed in control seedlings. The maximum reduction in H2O2 content was recorded in 10−8 M IAA. The decrease was about 23.33%, at 30DAS, 32.29%, at 45 DAS and 28.85% at 60 DAS, whereas minimum decline was recorded in 10−6 M IAA treatment, which was about 14.16%, 15.69%, and 15.23% compared to their respective controls at 30, 45, and 60 DAS respectively. The result was further validated by visualization of H2O2 in leaves of control and 10−8 M IAA supplied plants at 45 DAS (Fig. 7A, B) where a noticeable decline in H2O2 content was reported in the presence of IAA.

Fig. 7.

Fig. 7

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Data presented here show the Stereo micrographs of adaxial leaf surface of 45-day-old Brassica juncea (L.) cv. Varuna showing 3,3′-diaminobenzidine staining (superoxide anion content) under A control B 10−8 M IAA and C H2O2− content at various IAA doses (0, 10−10, 10−8 or 10−6 M)) at 30, 45, and 60 days stage of growth. Values of bars with different letter are significantly different, P < 0.05 Duncan’s multiple range test

O2− content

Superoxide anion accumulation decreased with the increasing concentration of IAA (Fig. 8C) and 10−8 M proved best. The maximum decline was about 12.97%, 19.97%, and 21.10%, whereas minimum decline was reported in 10−6 M IAA which was about 4.32%, 12.31%, and 8.44% at 30, 45, and 60 DAS respectively, over their respective controls. The findings was further validated by visualization of O2− in leaves of control and 10−8 M supplied plants at 45 DAS (Fig. 8A, B) where a marked decline in H2O2 content were observed in the presence of IAA.

Fig. 8.

Fig. 8

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Data presented here show the Stereo micrographs of adaxial leaf surface of 45-day-old Brassica juncea (L.) cv. Varuna showing nitrobluetetrazolium staining (superoxide anion content) under A control B 10−8 M IAA and c O2− content at various IAA doses (0, 10−10, 10−8 or 10−6 M) at 30, 45, and 60 days stage of growth. Values of bars with different letter are significantly different, P < 0.05 Duncan’s multiple range test

Localization of O2− and H2O2

The level of O2− was visualized by blue staining and H2O2 by brownish spots (Figs. 7A, B, 8A, B) on leaves. In control plants stained spots were more prominent over IAA sprayed plants. Also, the maximum reduction in the O2− and H2O2 accumulation was visualized in the leaves of plants treated with IAA (10−8 M).

Confocal microscopy

DCF-DA fluorescence in root is directly linked to ROS accumulation. Maximum fluorescent nuclei were reported in DDW treated control roots illustrating maximum ROS accumulation in comparison to IAA (10−8 M) treated roots (Fig. 9A, B). Fluorescent nucleic acid staining dye (propidium iodide) enters the dead cell membrane and reacts with nuclei and demonstrates cell death. Maximum the illuminating nuclei, lesser is the cell viability. Number of illuminating fluorescent nuclei declined in roots of the IAA (10–8) supplied plants over the control (Fig. 9C, D).

Fig. 9.

Fig. 9

Open in a new tab

Cell viability test (A, B) and ROS localization (C, D) in roots was performed on 45 days old roots of Brassica juncea and confocal microscopic images were obtained. Abundance of fluorescence stain (green) indicate more ROS accumulation A control and B 10−8 M IAA, and abundance of stained nuclei (red) is indicative of less cell viability C control and D 10−8 M IAA treated plants (color figure online)

Discussion

In this study, different doses of exogenous IAA were applied to investigate the changes in growth, physiological, and biochemical indicators of Brassica juncea. The growth attributes (root length, shoot length, root fresh mass, root dry mass, shoot fresh mass, shoot dry mass, and leaf area) are relevant biomarkers of growth and development and these growth biomarkers were significantly increased by all the applied concentrations of IAA and responses in a dose-dependent manner (Fig. 1A–F). The enhancement in growth biomarkers was previously reported in Vigna radiata (Ali et al. 2008), groundnut (Lee 1990), cotton (Kapgate et al. 1989), rice (Kaur and Singh 1987), and soybean (Sarkar et al. 2002). Hayat et al. (2006) also demonstrated the elevation of growth parameters in Solanum melongena when treated with different doses of IAA. This improvement may perhaps be due to its characteristic response to stimulate cell division, cell elongation, and cell differentiation (Sitbon and Perrot-rechenman 1997). Further, Aux increases the proton concentration in cells by stimulating H+ ATPase leading to acidification, which ultimately favors cell wall expansion required to drive cell division (Rayle and Cleland 1977; Cleland et al. 1977). Moreover, the stimulatory effect of Aux on growth is also contributed to its impact on enlarging leaf area and associated photosynthetic attributes (Fig. 3A–D; Naeem et al. 2004), increased cell multiplication, and higher production of carbohydrate content in plants (Sadak et al. 2013). Similar dose dependent relationship was reported by Kadiri (1999) in Capsicum annum L. plant where IAA application significantly increased the plant height, stem thickness, leaf area, leaf number, root and shoot biomass, chlorophyll content, and fruit yield. In agreement with our results, Ahmed et al. (2001) also found the same pattern of diminishing effects in growth at higher concentrations. The impact of IAA started fading with the application of higher dose of IAA (Fig. 1) possibly because it mediated a decrease in endogenous IAA concentrations by an apparent feedback mechanism (Ribnicky et al. 1996).

Figure 2D unfurls the positive impact of IAA on SPAD value over control plants. Our data were in concurrence with Kaya et al. (2013), as they reported the enhanced pigment composition in IAA supplied maize plants. This enhancement is attributed to the promotion of pigment synthesis which is evident from increasing magnesium (Mg) content (Table 1), the fundamental elements required for chlorophyll synthesis, and also by down-signaling the process of chlorophyll degradation (Jacobs 1979) in plants receiving Aux spray. Moreover, the scanning electron micrographs of leaf surface also divulge the IAA stimulated the increase of guard cells and adjacent epidermal cells leading to an increase in stomatal aperture as compared to untreated plants (Fig. 5). This increase in stomatal aperture is due to the stimulation of H+ ATPase by IAA, driving the influx of K+ into the guard cells (Takahashi et al 2012). Potassium K+ performs a vital role in regulating stomatal opening and closure (Siddiqui et al. 2018). Improved levels of K+ within and around the guard cells, helps to regulate stomatal opening and closure by modulating the solute potential inside and outside the guard cell to sustain wider open stomatal pore (Smith and Stewart 1990). Hence, the reported improvement in the stomatal pore (Fig. 5) may perhaps be due to improved concentration of K in cells (Fig. 6). In addition, Mg and K in their charged state regulate various enzymes required for photosynthesis and respiration processes (Taiz and Zeiger 1998). Therefore, the reported enhancement in K and Mg levels within the cells may possibly have improved chlorophyll biosynthesis and ultimately leads to an upsurge in photosynthetic activity in IAA applied plants. Another nutrient influenced by the exogenous treatment of IAA was Ca. In fact, all the applied IAA levels increased the Ca content of leaves (Fig. 6), significantly and 10−8 M IAA proved most effective among all the applied concentrations. This improvement might be associated to the counter-transport of IAA–Ca inside the plant cells, which directly stimulates H+-ATPase and leads to a decline in apoplastic pH (Marschner and OssenbergNeuhaus, 1977). Similarly, IAA regulated increase in the nutrient composition has previously been demonstrated by Vardhini et al. (2014).

Photosynthetic parameters (PN, gs, Ci, and E) were significantly increased at all the IAA concentrations applied (Fig. 3A–D). IAA mediated increase in stomatal conductance (gs), size of stomatal aperture and transpiration rate (E) increased the CO2 diffusion which is evident from higher Ci values (Fig. 3B–D). CA is the second most abundant protein in the chloroplast of plants (Okabe et al. 1984) and facilitates the transport of CO2 through the cell membrane and maintains the supply of CO2 to the RUBISCO essential for carbon fixation (Majeau and Coleman 1994). In the current experiment, all the tested concentrations of IAA significantly enhanced the CA levels (Fig. 4A). In support of our results, Pandey et al. (2000) also revealed the improved CA levels in Aux treated plants which directly increased the photosynthetic rate (Chaterjee et al. 1976) and elevated the levels of phosphorylation (Tamas et al. 1973). Hence, it could be suggested that the accumulation of CO2 concentration inside the cells resulted in its efficient binding with rubisco in the presence of CA (Fig. 4A) which marked the beginning of calvin cycle (Badger and Price 1994) and resulted in elevated PN (Fig. 3A). It has been reported earlier that improvement in gas exchange parameters is a result of enhanced activities of certain enzymes involved in photosynthesis (Ahmad et al. 2001) as well as elevated levels of phosphorylation (Chatterjee et al. 1976) in plants. Ali et al. (2008) also reported the enhanced levels of pigment composition and PN in presence of Aux in Vigna radiata. Moreover, phosphorus (P) and sulphur (S) are active constituents of various compounds like sugar-phosphate; phospholipids; amino acids and utilized in energy metabolism, and performs key role in production of proteins, ATP, ADP, and NADPH (Taiz and Zeiger 1998). Therefore, the reported enhancement in P content (Fig. 6) will also contribute to the enhanced growth and photosynthetic efficiency of IAA-sprayed plants.

NR is a key enzyme required for the conversion of nitrate to nitrite in the plant cells. Nitrate reduction is the basic step of nitrogen metabolism in plants. In our findings, all the tested IAA concentrations increased the NR activity in test plants (Fig. 3C) which signifies the increase in the rate of conversion of nitrate to nitrite by IAA. NR activity depends on the presence of hormones like Aux and its isoforms (Ahmad and Hayat 1999). Similar improvement in NR activity by exogenous treatment of IAA was previously demonstrated by Ali et al. (2008) in Vigna radiata and Ahmad et al. (2001) in Brassica juncea. And since, NR is involved in Aux-mediated nitric oxide (NO) induction in plants (Kolbert et al. 2008) the increase in growth and photosynthesis by Aux might follow an NO dependent pathway as the latter is known for enhancing PN and growth in mustard (Sami et al. 2020). The biosynthesis of proline in plants is linked to the assimilation of nitrogen. It is a low molecular weight compound and acts as non-enzymatic antioxidant in plants (Bhandari et al. 2017; Czarnocka and Karpinski 2018). In the present study, all the applied Aux concentrations increased the proline level significantly as compared to non-treated plants (Fig. 4B). This enhancement may perhaps be due to increased NR activity (Fig. 4C), which ultimately led to an increase in amino acid synthesis (Foyer et al. 2003). Further, the application of Aux also increases the proline content in plants as reported in garden pea (Sergiev et al. 2017).

Plant Growth Regulators are well recognized for their growth-promoting activities and increase crop productivity (Ammanullah et al. 2010). In the present experiment, we tested the different IAA concentrations (0, 10−10, 10−8 and 10−6 M) on Brassica juncea, and noted their beneficial role on primary biochemical metabolites as presented in (Fig. 3E, F). All the concentrations applied showed an increase in primary metabolites accumulation (protein, total soluble sugar, reducing sugars, and starch content). The enhancement is attributed to elevated photosynthetic activities (Fig. 3A–D), which results in increased growth of plants (Awan et al. 1999).In support of our findings, enhanced carbohydrate levels by IAA application were also reported in Vicia faba (Sadak et al. 2013). Moreover, IAA application increased the protein content in Pleurotus sajor-caju (Mukhopadhyay et al. 2005). The possible reason behind this might be the effect of plant growth regulators on the transcription and translational processes of protein synthesis, as reported in Cicer arietinum (Dhingra et al. 1994), and also regulate the quantity and allocation of assimilates in plants (Galston and Davier 1969).

An increase in the relative water content (RWC) was also detected in presence of Aux (Fig. 2F). The balance between the water loss and uptake determines the leaf water status in a plant wherein, the water loss is regulated by stomatal pore adjustments (Pantin et al. 2012; 2013). Leaf water potential declines when water loss exceeds the water uptake. Low water potential tends to form air bubbles within the xylem vessels, obstructing the water uptake (Perrone et al. 2012; Brodersen and McElrone 2013) however, recommencement of water supply, restores its uptake (Brodersen and McElrone 2013). In the present study, water loss occurred via transpiration but still the plant presented higher RWC values in Aux treated plants, suggesting that the water loss did not surpass its uptake. Additionally, the enhanced root growth (Fig. 1B, D, F) and confocal microscopic studies (comparatively low ROS and cell death; Fig. 9A–D) in presence of Aux manifested healthy root system which probably improved the water uptake by roots and maintained the stability between water uptake and its loss in the plant.

Superoxides are regularly produced in chloroplasts during chlorophyll biosynthesis and photosynthetic electron transport, and their generation is strictly associated with hydrogen peroxide (H2O2) generation (Slesak et al. 2007). In the present investigation, ROS production decreased in presence of Aux (Figs. 7, 8 and 9), which is attributed to the enhanced activity of antioxidant enzymes (Fig. 2). SOD is the primary enzyme that detoxifies the cellular environment including photosynthetic apparatus by converting superoxide ions to H2O2, which thereafter is reduced by CAT and POX (Pasternak et al.2002; Apel and Hirt 2004; Siddiqui et al. 2018) to water. Increased activities of antioxidant enzymes by Aux were also reported by Piotrowska-Niczyporuk and Bajguz (2014) in Chlorella vulgaris. The results suggest that Aux plays an important role in releasing the brake on photosynthesis (Fig. 3A) by limiting the ROS generation (Figs. 7 and 8) leading to enhanced growth and development of plants (Fig. 1).

Conclusion

The present findings conclude that the exogenous application of IAA proved effective in promoting the growth of Brassica juncea. Experimental evidence suggests that IAA enhanced the photosynthetic and antioxidant enzyme machinery leading to enhance the growth of the plant. Further IAA stabilized K concentration within and around cells to widen the stomatal aperture and enhanced the stomatal opening and conductance which resulted in enhanced CO2 influx inside the leaf cells. Moreover, elevated E resulted in better absorption of mineral elements, where increment in Mg content contributed to escalating chlorophyll biosynthesis upon IAA application. Moreover, it also enhanced the elemental composition required for optimal growth and photosynthesis. IAA spray proved effective in enhancing the photosynthetic attributes, carbohydrate, and protein contents. Besides, IAA promoted the activity of antioxidants which resulted in a significant reduction in ROS and lipid peroxidation in the plants thereby, enhancing cell viability. Our results clearly indicate that IAA acts as a potent plant growth regulator capable of modulating the physiology of plants.

Acknowledgements

The authors are thankful to the Aligarh Muslim University for providing all the required facilities during the experiment and Anayat Rasool Mir is also thankful to the University Grant Commission, New Delhi, India for the award of Non-Net Fellowship.

Compliance with ethical standards

Conflict of interest

Authors declare that there is no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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