The Efficiency of Humic Acid for Improving Salinity Tolerance in Salt Sensitive Rice (Oryza sativa): Growth Responses and Physiological Mechanisms

High-yielding rice cultivars exhibit a great performance in non-saline fields; however, their growth and productivity are greatly reduced in salt-affected lands. Humic acid has a promising stress-mitigating potential and can be effective in improving salt tolerance in salinity sensitive rice cultivars. Herein, seeds of Giza 177 (high-yielding but salt-sensitive rice cultivar) were primed in 40 mg/l humic acid, sown, and maintained. Then growth and physiological responses of the humic acid-primed plants to increased levels of salinity (EC: 0.55, 3.40, 6.77, and 8.00 mS/cm) were evaluated at the reproductive stage. Increasing salinity induced a progressive retardation in plant height, leaf area, fresh and dry weights. Such retardation was associated with Na+ buildup in shoot and root, high electrolyte leakage and accumulation of malondialdehyde, total soluble sugars, sucrose, glucose, proline, total soluble proteins, flavonoids, and phenolics. In contrast, salinity reduced K+, K+/Na+ ratio, total carbohydrates, and the activity of catalase, peroxidase, and polyphenol oxidase. Humic acid enhanced growth under non-saline and saline conditions. The humic acid-induced improvement in salt tolerance was associated with the reduction of Na+ toxicity, increasing K+/Na+ ratio, regulating osmolytes concentration, and enhancing the activities of antioxidant enzymes and thus reduce the oxidative stress. These results indicate that humic acid successfully reduced the salinity-induced plant damage, improved metabolism, and maintained active growth of Giza 177 under saline irrigation.


Introduction
Rice (Oryza sativa L.) is one of the most important economic cereals worldwide.Its production worldwide approached 512.6 million tons in 2022 (FAO 2022).Rice is wildly cultivated in the six continents, represents an essential source of food and energy for more than 50% of the world's population, and is an important source of employment for rural people (Lou et al. 2012;Hoang et al. 2016).It has high nutritional value of carbohydrate, protein, fat, fiber, vitamins, minerals, and numerous bioactive compounds (Verma and Srivastav 2020).
Rice growth and productivity are very sensitive to salinity.In fact, rice is classified as the most sensitive cereal with a salinity tolerance threshold of only 3 dSm -1 for most cultivated rice varieties.Its grain yield is reduced by 10% at 3.5 dSm -1 and by 50% at 7.2 dSm -1 (Umali 1993;Das et al. 2015;Thorat et al. 2018).Unfortunately, the sea level rises, and this phenomenon is expected to continue with the ongoing global warming which will increase the hazard of salinity in many irrigated lands (IPCC 2018).In fact, 50% of these lands are expected to suffer from salt stress by 2050 which will form a major challenge for the world rice production (Hossain 2019).Therefore, it is critical to better understand the salinity-induced retardation in rice growth and productivity and use this knowledge to identify potential strategies for enhancing tolerance against salinity in the high-yielding rice cultivars of this important crop.
Salinity negatively impacts rice growth and grain yield through different mechanisms (Shahzad et al. 2017;Lekklar et al. 2019).It induces various types of stresses such as ionic, osmotic, and oxidative stresses.Ionic stress develops as a result of increased Na + and Cl -ion accumulation in plant tissues, whereas osmotic stress appears because of the reduction in the plant's capacity to absorb water (Parihar et al. 2015).The oxidative stress in triggered by the salinity-induced generation of reactive oxygen species (ROS), which occurs as a result of the salinity-induced reduction in stomatal conductance, interference with the photosystems and photosynthetic enzymes (Hasanuzzaman et al. 2018).Altogether, these salinity-induced stresses interrupt metabolic processes, deteriorate cellular organelles, and can induce cell death (Munns and Tester 2008;Amirjani 2011;Kanojia and Dijkwel 2018).In response to salinity stress, plants regulate Na + and Cl -uptake to avoid ionic toxicity and activate various defense mechanisms including biosynthesis of osmo-protectants, ion homeostasis and compartmentation of toxic ions, production of enzymic and nonenzymic antioxidants, and activation of programmed cell death (Munns and Gilliham 2015;Hoang et al. 2016;Chen et al. 2021).
Seed priming with stress-alleviating chemicals has been one of the effective strategies to activate mechanisms of salt tolerance in the primed plants (Ellouzi et al. 2013;Ibrahim 2016;Johnson and Puthur 2021).Various priming compounds exert positive effects on rice germination, growth, and biochemical qualities such as high content of protein, carbohydrates, and antioxidants (Hussain et al. 2017;Du et al. 2019).Humic acid is one of the promising priming factors and its ability to improve plant development and tolerance to abiotic stress has been tried in many crops (Çimrin et al. 2010;Aydin et al. 2012;Sheteiwy et al. 2017).Humic acid is a component of humic substances that are derived from breakdown of the biological remnants is soil.It is soluble at high pH.It positively impacts soil water retention and storing soil cations because of its negative charge, making them available to plants and reducing their leaching from soil.It also has a hormone-like activity (De Melo et al. 2016;Yang et al. 2021).In addition, humic acid has a potential to activate several metabolic processes such as respiration, photosynthesis, mineral nutrient uptake and transcriptional activation (Trevisan et al. 2010).
Giza 177 is an early maturing and high yielding rice cultivar with excellent cooking and eating qualities.How-ever, it's growth and yield are significantly reduced in saltaffected lands and consequently it is classified as a salinity sensitive rice cultivar (Elmoghazy and Elshenawy 2018;Badawy et al. 2021).The physiological processes underlying its sensitivity to salinity are not well known.In addition, the possible improvement of its salinity tolerance by priming with humic acid has not been reported.Here, we hypothesize that priming Giza 177 seeds in humic acid can increase its tolerance against salinity and maintains its active growth in salt-affected lands.Therefore, the main objective of the current study was to investigate the growth and physiological responses of Giza 177 under increased levels of salinity in absence and presence of humic acid at reproductive stage-the most vulnerable stage in rice plant's life cycle to salinity stress (Ahmadizadeh et al. 2016;Singh et al. 2021).

Plant Material and Experimental Setup
Rice (Oryza sativa L. cv.Giza 177) seeds were obtained from Rice Research and Training Center in Sakha, Kafr El-Sheik, Egypt.The seeds were surface sterilized using 3.6% (v/v) sodium hypochlorite for 15 min and washed thrice with sterilized distilled water.Seeds were then allocated into two groups: the first group was soaked in distilled water as a control whereas the other group was soaked in 40 mg/l humic acids for 72 h at 27 ± 2 °C in the dark.Each of the control and humic acid-treated seeds with emerging radicals were carefully sown in 10 pots (25 cm diameter, with 7 kg soil), germinated, and maintained in a greenhouse for 28 days (representing the nursery stage).The uniformly rice plantlets were then transplanted into 40 larger pots (30 cm in diameter, 30 cm height, with 10 kg soil, 15 plantlet/pot, 20 pots each treatment).Plantlets were maintained in the greenhouse (12-hour light/12-hour dark cycle, ~75% relative humidity, and 450 µmol m -2 s -1 light intensity) until the complete recovery and establishment.

Stress Application, Plant Maintaining, and Sample Collection
Homogeneous recovered plantlets were thinned to 10 plantlets/pot and used for the salinity stress application.Pots with homogeneous recovered controls and humic acid-treated rice plants were allocated into four subgroups.Salinity stress was imposed by irrigation with saline water of 5, 10, and 12.5% seawater dilutions, which are equivalent to EC values of 3.40, 6.77, and 8.00 mS/cm, respectively.One subgroup of the 28-old plantlets was irrigated with tap water (0.55 mS/cm) and used as a control.Every four K Plants were maintained in the greenhouse till reproductive stage (75 days after transplantation), then the whole plants were uprooted, washed thoroughly with distilled water, separated into roots and shoots, and processed for downstream analysis.Depending on the downstream physiological analysis, fresh plant samples were either immediately dipped in liquid N and stored at -80 °C for malondialdehyde (MDA), protein content, and enzyme analysis, or used for measuring growth traits, oven dried at 65 °C to a constant dry weight and used for elemental and other biochemical analyses.

Growth Analysis
Root length was measured from the point of root/shoot junction to the tip of the longest root.Shoot length was measured from the point of root/shoot junction to the tip of the longest leaf.Fresh and dry weights were recorded using digital balance.Leaf area was determined using the equation (Leaf area = length × breadth × 0.75) (Palaniswamy and Gomez 1974).The salt tolerance index (STI) was calculated as the ratio of the total dry weight of salinity-stressed Giza 177 plants relative to the total dry weight of the control plants (Tao et al. 2021).

Elemental Analysis
Dry shoot and root samples from each treatment were ground into homogenous fine powder using a stainlesssteel grinder.Known weights of the dried powdered tissues were digested in 5 ml HNO3 and 1 ml of perchloric acid according to the method of Motsara and Roy (2008).Elemental analysis of Na + and K + was performed using Flame photometer (PFP7, Jenway) and expressed as mmol g -1 DWT.

Estimation of Carbohydrates
Known weights of the dried plant samples were extracted in 80% (v/v) ethanol at 25 °C as described previously (Yemm and Willis 1954;Li et al. 2015).The ethanolic extracts were used for spectrophotometric determination of total soluble sugars (TSS) and sucrose using anthrone reagent at 625 nm (Yemm and Willis 1954;Ejaz et al. 2022) and 620 nm ( Van Handel 1968), respectively.Also, glucose was determined spectrophotometrically using O-toluidine reagent at 625 nm (Feteris 1965;Riazi et al. 1985).Total carbohydrates (TC) were extracted by mixing of 0.1 g of dry tissues with 5 ml of HCl (2.5 N) in a boiling water bath for 3 h then neutralized using Na2CO3 after cooling to room temperature.The TC in the extract was then determined spectrophotometrically by anthrone reagent at 630 nm (Hedge and Hofreiter 1962).

Estimation of Protein
Known wights of the frozen ground tissues were extracted in Tris-HCl buffer as described by Scarponi and Perucci (1986) and the total soluble proteins (TSP) in the extracts were determined spectrophotometrically at 595 nm using the dye-binding assay (Coomassie Brilliant Blue G250) (Bradford 1976).The concentration of TSP was calculated from a bovine serum albumin standard curve and expressed as mg protein g -1 FWT.

Determination of Proline
Known wights of dried plant samples were extracted in distilled water as described previously (Costa et al. 2011;Meychik et al. 2013) and the aqueous extracts were used for the spectrophotometric determination of proline using ninhydrin reagent at 520 nm (Bates et al. 1973).Proline was calculated using a standard curve and expressed as mg g -1 DWT.

Assessing the Oxidative Damage
Electrolyte leakage (EL) was assessed according to Shi et al. (2006) by incubating discs of freshly harvested plant leaves in 30 ml de-ionized water for 24 h in the dark at room temperature, then recording the initial electric conductivity (EC1) using EC meter (HANNA Instrument, HI 8033).The tubes were heated at 95 °C for 20 min, cooled down, and the final electrical conductivity (EC2) was measured.EL was then calculated as EL% = (EC1 / EC2) × 100.
Lipid peroxidation in frozen plant samples was monitored via determination of malondialdehyde (MDA) content (Heath and Packer 1968).Known weights of frozen leaf tissue were homogenized in 0.1% (w/v) trichloroacetic acid (TCA) at 4 °C and centrifuged at 12,000 rpm for 5 min.One ml of the supernatant with mixed with 4 ml of thiobarbituric acid reagent, heated at 90 °C for 30 min, and cooled.The mixtures were recentrifuged at 10,000 rpm for 5 min and the absorbance of the developed color was measured at 532 and 600 nm using spectrophotometer (Shimadzu model UV-160A).The measurements at A600 were subtracted from those at A532 and the MDA concentration was determined using an extension coefficient of 155 × 10 -3 µM -1 cm -1 and expressed as µmol g -1 FWT.

Determination of Non-Enzymatic Antioxidant Compounds
Total phenolics and flavonoids were extracted in methanol as described by Kosem et al. (2007).Total phenols in the methanolic extracts were determined spectrophotometrically (Shimadzu, model UV-160A) using Folin-Ciocalteu reagent at 765 nm.The concentration of total phenolics were then calculated from a gallic acid standard curve and expressed as mg of gallic acid equivalents (GAE) g -1 DWT (Singleton and Rossi 1965).Flavonoids in the methanolic extracts were determined spectrophotometrically using AlCl3 method at 510 nm (Marinova et al. 2005).Flavonoid concentration was then calculated using quercetin standard curve and expressed as mg quercetin equivalent (QUE) g -1 DWT.

Antioxidant enzymes including (catalase [CAT], Peroxidase
[POX], and polyphenol oxidase [PPO]) were extracted by homogenizing known weights of frozen leaf samples in cold phosphate buffer (0.02 M, pH 7).The homogenates were centrifuged at 10,000 rpm and 4 °C for 20 min and the supernatants were collected and used as enzyme extracts (Agarwal and Shaheen 2007).CAT activity was monitored as described by Sinha (1972).Aliquots of 0.5 ml enzyme extracts were mixed with 1 ml of phosphate buffer (0.01 M, pH 7.0), 0.5 ml of H2O2 (0.2 M), and 0.4 ml of dist.H2O.The reaction mixtures were incubated at room temperature for 1 min and the enzymic reactions were stopped by adding 2 ml of the acid reagent (dichromate/acetic acid mixture; 1/3 v/v).The enzymatic reactions were heated for 10 min, cooled, and the developed color was measured at 610 nm using a spectrophotometer (Shimadzu model UV-160A).CAT activity was expressed as mM H2O2 consumed min -1 g -1 FWT.POX and PPO were assayed spectrophotometrically at 420 nm as described by Devi (2002).For POX, the reaction mixture contained 0.1 ml of enzyme extract, 3 ml of pyrogallol (0.05 M) prepared in phosphate buffer (0.1 M, pH 6), and 0.5 ml of H2O2 (1%).The mixture was mixed well and incubated for one minute at 25 °C.The POX enzymatic reaction was stopped with 1 ml of H2SO4 (2.5 N).One POX enzyme unit was defined as a unit min -1 g -1 FWT.PPO was measured by mixing 1 ml of enzyme extract, 1 ml of pyrogallol (0.1 M), and 2 ml of phosphate buffer (0.02 M, pH 7) for one minute at 25 °C.The enzymic reaction was stopped by adding 1 ml of H2SO4 (2.5 N).One PPO enzyme unit was defined as a unit min -1 g -1 FWT.

Statistical Analysis
The data were statistically analyzed by one-way analysis of variance (ANOVA) using statistical software CoStat Version 6.3.Data are presented as means ± SE and were compared using the Post Hoc Duncan's test at P < 0.05.Data were subjected to principal component analysis (PCA) and Pearson correlation coefficient using JMP Pro software.

Growth Responses of Giza 177 to Salinity and Humic Acid
Salinity stress decreased plant height, fresh weight, dry weight, and leaf area in a dose-dependent manner.These traits were severely affected at 12.5% seawater with maximum growth inhibition of 34.4, 76.6, 79.3, and 33.4%, respectively (Table 2).On the other hand, humic acid-treated rice plants showed better growth under saline and nonsaline conditions, compared to humic acid-untreated rice plants.The percentages of growth improvement in humic acid-treated stressed plants were 6.9, 9.0, and 10.2% in plant height, 57.7, 52.8, and 48.7% in plant fresh weight, and 47.6, 84.8, and 67.4% in plant dry weight at 5, 10, and 12.5% seawater, respectively.In leaf area, the corresponding percentages of the humic acid-induced increases were 32.8, 20.8, and 23.4%, respectively (Table 2).The salinity stressed Giza 177 plants showed significant differences in STI in the absence and presence of humic acid.In the humic acid-untreated stressed plants, the STI ranged from 0.21 to 0.54 whereas the corresponding values of the STI in the humic acid-treated stressed plants were significantly higher and fluctuated from 0.29 to 0.66 (Table 2).

Changes in Cellular K + and Na + in Response to Salinity and Humic Acid
The effect of salinity and humic acid on Na + , K + , and the K + /Na + ratio in Giza 177 shoots and roots are shown in Table 3. Salinity stress increased Na + concentration and simultaneously reduced the cellular K + in shoots and roots tissues.Such responses were translated into significant reduction in K + /Na + ratio in shoots and roots with 21.8, 44.8, and 49.9% reduction in shoots compared to 30.3, 39.4, and 49.5% in roots at 5, 10, and 12.5% seawater, respectively.Priming Giza 177 seeds with humic acid alleviated the Values are the means ± SE of three biological replicates.K + and Na + ions are expressed as mmol g -1 DWT.Different letters in each column show significant differences at P < 0.05, using the Post Hoc Duncan's test salinity-induced increases in the cellular concentration of Na + in shoots and roots when compared to humic acid-untreated rice plants.On the other hand, humic acid priming had insignificant effect on K + concentration in shoots and roots, instead, it increased the K + /Na + ratio by 2.2, 0.9, 25.4, and 9.8% in shoots and 15.6, 5.3, 3.7, and 2.1% in roots at 0, 5, 10, and 12.5% seawater, respectively.

Changes in Oxidative Stress in Response to Salinity and Humic Acid
To assesses the salinity-induced oxidative damages at 5, 10, and 12.5% seawater, MDA content and EL percentage of rice shoots were measured (Fig. 1).Salinity stress increased MDA content and EL percentage, with the highest percentage increases of 23.6 and 84.6% at 12.5% seawater, respectively.Priming Giza 177 seeds with humic acid significantly decreased the salinity-induced increases in MDA by 7.8, 6.8, and 7.6% compared to 14.7, 19.1, and 18.9% in EL at 5, 10, and 12.5% seawater, respectively.

Changes in Carbohydrates in Response to Salinity and Humic Acid
Compared to the control plants, salinity stress increased the content of TSS, sucrose, and glucose, however, it decreased the content of TC in rice shoots (Fig. 2).The highest increment was observed at 12.5% seawater with percentages of 59.0, 191.9, and 56.3% in TSS, sucrose, and glucose, respectively.Also, 12.5% seawater induced the greatest reduction in TC with a percentage of 21.9% when compared to the control plants.Under saline and non-saline conditions, humic acid-treated plants had significantly lower levels of TSS, sucrose, and glucose than humic acid-untreated plants.In contrast, humic acid priming significantly increased the accumulation of TC in salinity-unstressed plants by 5.9% compared to 6.3, 14.5, and 18.5% at 5, 10, and 12.5% seawater in salinity-stressed plants, respectively.

Changes in Protein and Proline in Response to Salinity and Humic Acid
The changes in total soluble proteins and proline content in Giza 177 plants are presented in Fig. 3. Compared to the control plants, increasing salinity level progressively increased the total soluble proteins and proline content.The highest percentages of increase in these two analytes were induced by 12.5% seawater, which caused inductions of 59.1% in total soluble proteins and 88.1% in proline content.Priming Giza 177 seeds with humic acid increased total soluble proteins in both the salinity-unstressed and salinity-stressed plants compared to the humic acid-untreated plants.In the salinity-unstressed plants, humic acid induced an increase of 18.8% in total soluble protein compared to increases of 11.0, 6.8, and 6.0% at 5, 10, and 12.5% seawater K Fig. 3 Effect of seed priming with humic acid (40 mg/l) on a total soluble proteins and b proline content of Giza 177 plants grown under increased levels of seawater (Control, 5%, 10%, 12.5%).Values are the means ± SE of three biological replicates.Different bar letters show significant differences at P < 0.05, using the Post Hoc Duncan's test in salinity-stressed plants, respectively.In contrast, priming Giza 177 seeds with humic acid showed no statistical effects on proline concentration in the salinity-unstressed and 5% stressed plants.However, compared to the humic aciduntreated stressed plants, humic acid priming significantly reduced the proline concentration in the salinity-stressed plants by 7.0 and 20.9% at 10 and 12.5% seawater levels, respectively.

Changes in Antioxidant System in Response to Salinity and Humic Acid
Increasing salinity level progressively increased the cellular concentrations of flavonoids and total phenols in Giza 177 plants with a maximum induction at 12.5% seawater (Fig. 4a, b).Seed priming with humic acid significantly decreased the content of these secondary antioxidants under non-saline and saline conditions.On the other hand, the antioxidant enzymes CAT, POX, and PPO showed varied responses in saline and non-saline treatments (Fig. 4c-e).Salinity stress was associated with a significant decrease in the activities of CAT, POX, and PPO with maximum reductions of 43.3, 17.8, and 26.7% in the severely salt-stressed Giza 177 rice plants (12.5% seawater), respectively.Seed priming with humic acid increased CAT, POX, and PPO activities in the humic acid-treated plants compared to humic acid-untreated plants under nonsaline and saline conditions.Such humic acid-induced increases in CAT, POX, and PPO in salinity-unstressed plants approached 14.4, 13.4, and 2.8%, respectively.The corresponding increases in the salinity-stressed plants at 5, 10 and 12.5% seawater approached 60.4,85.5,and 55.9% in CAT,32.0,17.1,and 12.1% in POX,and 52.2,37.4 and 37.5% in PPO.

Principal Component Analysis and Pearson Correlations
To test the relationships among the tested parameters and summarize the responses of Giza 177 under the applied treatments, the obtained data were subjected to the principal component analysis (PCA) (Fig. 5) and Pearson correlations (Fig. 6).The first principal component (PC1) accounted for 81% of the variance in the obtained data whereas the second component (PC2) captured 11.3% of the variance under different treatments (Fig. 5a).Shoots and roots Na + content, oxidative stress markers (MDA content and EL percentage), osmolytes (glucose, sucrose, TSS, and proline contents), and non-enzymatic antioxidant (flavonoids and total phenols) were mostly associated with C + 12.5% seawater, whereas total soluble proteins was mostly associated with H + 12.5% seawater.Growth parameters (plant height, plant fresh weight, plant dry weight, and leaf area), TC, shoots, and roots K + /Na + ratio were mostly associated with humic acid-treated plants under normal conditions (Humic), whereas antioxidant enzymes (CAT, POX, and PPO) were mostly associated with H + 5% seawater (Fig. 5c).In Fig. 6, heatmap correlation analysis showed many strong positive and negative correlations between Giza 177 growth and the tested parameters.For instance, strong positive relationships were observed between growth indices and roots K + (r 0.83), shoot K + /Na + ratio (r 0.94), root K + /Na + (r 0.93), TC (r 0.87), and antioxidant enzymes (CAT; r 0.76, POX; r 0.82, PPO; r 0.63).In contrast, strong negative relationships were observed between growth parameters and shoot Na + (r -0.92), root Na + (r -0.91), EL (r -0.95), and MDA (r -0.97) (Supplementary Fig. 1).
Effect of seed priming with humic acid (40 mg/l) on antioxidant activities a total flavonoids, b Total phenols, c catalase activity (CAT), d peroxidase activity (POX), and e polyphenol oxidase (PPO) of Giza 177 plants grown under increased levels of seawater (Control, 5%, 10%, 12.5%).Values are the means ± SE of three biological replicates.Different bar letters show significant differences at P < 0.05, using the Post Hoc Duncan's test

Discussion
In the current study, increasing the intensity of salinity stress progressively decreased all of the tested growth indices of Giza 177 rice plants.These results are consistent with the salinity-induced growth retardation of various rice varieties in many studies (Zhang et al. 2012;Ologundudu et al. 2014;Badawy et al. 2021).The adverse effects of salinity are attributed to the collective effects of its associated ionic, osmotic, and oxidative stresses which interrupt the plant's water status and metabolism and consequently hinders plant growth and development (Munns and Tester 2008;Rahman et al. 2016;Singh et al. 2018).In addition, salinity and its associated stresses lower the amount of chlorophyll which reduces photoassimilates content and reduces the activity of plant meristem (Abdallah et al. 2016;Kordrostami et al. 2017).Further, significant amounts of these photoassimilates are diverted away from supporting growth and are shunted toward synthesis of compactible compounds and antioxidants which also explain the reduced growth of salinity-stressed Giza 177 plants (Caretto et al. 2015;Munns and Gilliham 2015;Munns et al. 2020).The K Fig. 5 Principal component analysis (PCA) of growth, physiological, biochemical, and mineral traits in the investigated humic acid (40 mg/l) treated and non-treated Giza 177 plants grown under increased levels of seawater (Control, 5%, 10%, 12.5%).a PCA loading plot of 23 measured traits, b PCA score plot, and c PCA biplot above salinity-induced deteriorative effects on Giza 177 growth were significantly alleviated by priming its seeds with humic acid.Humic acid also improved the STI of the salinity-stressed Giza 177 plants (Table 2) which is consistent with the reported beneficial effects of humic acid on STI in sorghum (Ali et al. 2020) and strawberry (Saidimoradi et al. 2019).Such humic acid-induced increase in STI is attributed to its positive impact on biomass accumulation and the K + /Na + ratio.The above growth responses are supported by the PCA biplot, which demonstrated a substantial correlation between growth traits and humic acid treatment under normal conditions (Humic).These results coincide with the positive effects of humic acid on growth of maize (Kaya et al. 2018) and soybean seedlings (Matuszak-Slamani et al. 2017).It has been reported that humic acid induces biochemical changes in membranes and cytoplasmic components that support plant development and salt tolerance (Ouni et al. 2014).In addition, humic acid also enhanced maize growth by increasing cell division which was associated with the induction of antioxidant enzyme activity and ATPase expression (Morozesk et al. 2017).
The above salinity-induced retardation in Giza 177 shoots and roots growth was associated with a Na + buildup and a reduction in both K + levels and the K + /Na + ratio.In Heatmap of Pearson correlation analysis of all traits in humic acid (40 mg/l) treated and non-treated Giza 177 plants grown under increased levels of seawater (Control, 5%, 10%, 12.5%).Red and blue color represent positive and negative correlations, respectively, according to the color scale fact, the salinity-induced Na + influx has been considered as the main reason underlaying the observed reduction K + and the K + /Na + ratio in shoots and roots of other rice varieties (Rahman et al. 2016).Along with lowering K + levels, Na + buildup also hampers the uptake of macronutrients such as Ca 2+ and Mg 2+ , resulting in ion imbalance and plant growth retardation (Akter and Oue 2018).In this regard, our heatmap correlation analysis revealed a strong inverse correlation between growth traits and the Na + content of shoots and roots.Seed priming with humic acid reduced the amount of Na + in the shoots and roots and consequently increased the K + /Na + ratio.Such humic acid-induced reduction of the Na + ions in shoots and roots has been reported in wheat (Abbas et al. 2022) and barley plants (Jarošová et al. 2016).These effects of humic acid mimic the behavior of the salinity tolerant rice cultivar (Pokkali) which avoids salinity stress by minimizing its cellular Na + under saline irrigation (Akter and Oue 2018).The PCA biplot showed that the Na + contents of shoots and roots were mostly associated with C + 12.5% seawater whereas the K + /Na + ratio in shoots and roots were mostly associated with humic acidtreated plants under normal conditions (Humic).
The salinity-stressed Giza 177 rice plants suffered from a significant oxidative damage as indicated by the high EL percentage and MDA content in their leaves.These findings are consistent with the reported accumulation of ROS in salinity-stressed wheat cultivars, which increases EL percentage and MDA content, thus reducing membrane stability (Kaur et al. 2017).Our results also agree with the reported salinity-induced disruption of the balance between both ROS production and scavenging which increases the cellular level of ROS and causes severe oxidative damages to numerous cellular components particularly in salinity sensitive plants (Choudhury et al. 2017;Sachdev et al. 2021).Such responses of Giza 177 are thus coincided with its classification as a salinity sensitive variety by rice breeders (Badawy et al. 2021).Priming Giza 177 seeds with humic acid reduced the oxidative stress the plants encounter as evidenced by their low EL percentage and MDA content.Similar humic acid suppressive effects on EL and MDA content have been reported in salinity-stressed maize cultivars (Kaya et al. 2018).Interestingly, PCA biplot and heatmap correlation analysis showed strong association among the shoots and roots Na + , EL percentage, and MDA content.
The salinity-stressed Giza 177 plants accumulated significant amounts of glucose, sucrose, TSS, proline, and total soluble proteins, but they reduced TC level.Interestingly, the heatmap correlation analysis revealed that the Na + contents in Giza 177 shoots and roots was positively correlated to the above compatible organic solutes, while negatively correlated with TC.The accumulation of the above organic solutes coincides with their active contribution to the maintenance of cell osmotic potential, membrane stability, and antioxidant defense systems (Abdelrahman et al. 2018;Mushtaq et al. 2020).It worth mentioning that the osmotic adjustment with organic solutes consumes energy, therefore the successful responses of salinity-stressed plants most likely involve a "cross-talk" between allocation of available resources toward either synthesis of these compatible compounds or toward growth (Munns et al. 2020).Priming Giza 177 seeds with humic acid modified the osmolyte content by reducing the salinity-induced accumulation of glucose, sucrose, TSS, and proline.In contrast, humic acid increased the levels of total soluble proteins and TC.Similar promotive effects of humic acid on protein synthesis have been reported in sorghum (Ali et al. 2020) and almond rootstocks (Hatami et al. 2018).These results appeared more clearly in PCA biplot which showed a strong association of shoots Na + , roots Na + , glucose, sucrose, TSS, and proline with C + 12.5%, total soluble proteins with H + 12.5%, and TC with humic acid-treated plants under normal conditions (Humic).These responses along with the above humic acid-improved growth under salinity stress suggest that humic acid alleviated the salinityinduced damage in Giza 177 plants.Therefore, in humic acid-treated plants, the reduced level of glucose, sucrose, TSS, and proline as well as the elevated level of TC and total soluble protein may be a strategy to secure more carbon to support better growth under salinity.Consistent with that, alleviating the salinity-induced harmful effects on cotton plants by humic acid has been attributed to its induced reduction of TSS and proline in leaves (Rady et al. 2016).In addition, reduction of the salinity-induced proline buildup K by acid in pepper (Yildiztekin et al. 2018) and maize (Kaya et al. 2018) plants has been reported.Higher concentration of proline may be a symptom of plant damage, even though it is required for plants to withstand salt stress.Additionally, high proline concentrations may have negative effects on plant growth and metabolism (Hayat et al. 2012;Mazhar et al. 2012).The mechanisms of the toxic effects of high concentrations of proline on plant growth in stressful environment is not yet clear.However, high concentration of proline may negatively interfere with DNA stability via destabilization of the DNA helix, increasing both the susceptibility and insensitivity of DNA to SI nuclease and DNAase1, respectively.In addition, high levels of proline may induce feedback inhibition of 1-pyroline-5-carboxylate synthetase which indirectly inhibits organogenesis (Hayat et al. 2012).
The salinity-stressed Giza 177 rice cultivar accumulated significantly higher total phenolic and flavonoids antioxidants in their leaves than their control plants.However, they exhibited significantly lower activities of antioxidant enzymes (CAT, POX, and PPO).The salinity-induced accumulation of phenolic and flavonoids are consistent with their efficiency in ROS scavenging due to their high reactivity as hydrogen or electron donors to stabilize the unpaired electron (Mathew et al. 2015;Dumanović et al. 2021;Liu et al. 2022).The salinity-induced inhibition in antioxidant enzymes in Giza 177 is consistent with the reported lower activity of antioxidant enzymes (CAT and SOD) in the salt-sensitive BRRI dhan49 rice cultivar (Hasanuzzaman et al. 2014).Such response can be attributed to alteration in enzyme subunits assembly or ineffective synthesis of the enzyme under salinity (Hasanuzzaman et al. 2014) or to the prolonged salinity-induced severe oxidative damage (Hu et al. 2011;Paul et al. 2017).Seed priming with humic acid increased the activities of antioxidant enzymes (CAT, POX, and PPO) at low salinity levels but decreased them at the highest salinity level (H + 12.5% seawater) when compared to humic acid-treated plants under normal conditions (Humic).It also decreased the content of non-enzymatic antioxidants (total phenols and flavonoids) in the leaves under non-saline and saline conditions when compared to the humic acid-untreated plants.These findings are consistent with the reported ability of humic acid-induced reduction in total phenolic content in arsenic-stressed rice (Mridha et al. 2021) and salinity-stressed Vitex trifolia plant (Ashour et al. 2021).The PCA analysis revealed a strong correlation of growth parameters with humic acid-treated plants under normal conditions (Humic), antioxidant enzymes (CAT, POX, and PPO) with H + 5% seawater, and non-enzymatic antioxidants (total phenols and flavonoids) with C + 12.5% seawater.These findings demonstrate the beneficial effects of humic acid on rice growth and salinity stress tolerance.
In the current study, the obtained humic acid-induced growth and physiological responses indicate the effectiveness of humic acid in mitigating the salinity-induced disruption of the ionic balance, alteration in osmolyte production, and oxidative stress.These findings support its promising potential for maintaining active growth and improving crop salt tolerance in salt-affected lands.

Conclusion
The high-yielding Giza 177 rice cultivar is highly sensitive to salinity.Its growth is greatly retarded by salinity and such retardation is associated with Na + buildup in the shoots and roots, induction of electrolyte leakage, and accumulation of MDA, glucose, sucrose, TSS, proline, total soluble proteins, and non-enzymic antioxidants.In contrast, the salinity-induced growth retardation of Giza 177 is also associated with a reduction in K + , K + /Na + ratio, TC, and activities of antioxidant enzymes.Priming Giza 177 seeds with humic acid (40 mg/l) alleviated the salinity-induced deteriorative effects and improved its growth.The humic acid-improved growth was associated with reduction of Na + toxicity in shoots and roots, induction of antioxidant enzymes including CAT, POX, and PPO and active tradingoff of the available resources between growth and synthesis of compatible solutes and antioxidants (Fig. 7).Therefore, priming Giza 177 seeds with humic acid is a useful strategy for increasing its salinity tolerance.The reported promotive Fig. 7 A schematic model of the mechanisms of humic acid-induced improvement of salt tolerance and maintaining active growth in the Giza 177 rice cultivar.The red and green arrows denote inducing effects whereas blunt-end arrows depict inhibiting effects of salinity and humic acid effects in the current study may be also other crops and improve their growth and in salt-affected lands.

Fig. 2
Fig. 2 Effect of seed priming with humic acid (40 mg/l) on a glucose, b sucrose, c total soluble sugars (TSS), and d total carbohydrates (TC) of Giza 177 plants grown under increased levels of seawater (Control, 5%, 10%, 12.5%).Values are the means ± SE of three biological replicates.Different bar letters show significant differences at P < 0.05, using the Post Hoc Duncan's test

Table 1
The treatments used in the current study

Table 2
Effects of seed priming with humic acid (40 mg/l) on growth parameters and salt tolerance index (STI) of Giza 177 plants grown under increased levels of seawater (Control, 5%, 10%, 12.5%) Values are the means ± SE of seven biological replicates.Different letters in each column show significant differences at P < 0.05, using the Post Hoc Duncan's test

Table 3
Effects of seed priming with humic acid (40 mg/l) on ionic contents of shoots and roots of Giza 177 plants grown under increased levels of seawater (Control, 5%, 10%, 12.5%) Physiological effects of brown seaweed (Ascophyllum nodosum) and humic substances on plant growth, enzyme activities of certain pepper plants grown under salt stress.Acta Biol Hung 69:325-335.https://doi.org/10.1556/018.68.2018.3.8Zhang ZH, Liu Q, Song HX, Rong XM, Abdelbagi MI (2012) Responses of different rice (Oryza sativa L.) genotypes to salt stress and relation to carbohydrate metabolism and chlorophyll content.Afr J Agric Res 7:19-27.https://doi.org/10.5897/ajar11.834Mohamed E. Abu-Ria has MSc in Plant Physiology and is currently works as an assistant lecturer of plant physiology in Botany Department, Faculty of Science, Mansoura University, Egypt.He is interested in dissection of plant physiological responses under abiotic stress.His research attracted funds from the Egyptian Science, Technology, and Innovation Funding Authority (STDF) under grant number 44499.