Unlocking NUE Potential via PASP-Ca Synergist: Insights into physio-biochemical, enzymatic and molecular analyses of contrasting potato genotypes in aeroponics

The synergistic effect of various PASP-Ca treatments on morphophysiological, N-related, and enzymatic parameters 25 coupled with their transcript levels (shoot and root) in four potato genotypes having contrasting NUEs under low and 26 high N supplies in aeroponics.


Introduction
The phenomenal growth in fertilizer use, particularly nitrogen, is the swiftest method for improving crop production to meet the growing global population's food demand, which is expected to be doubled by 2030 (Sahay et al., 2021).
However, N recovery is less than 50%, leading to N loss coupled with poor nitrogen-use efficiency (NUE), increasingly causing negative repercussions to human health and the ecosystem (Hsieh et al., 2018;Sagwal et al., 2023).To address this issue, enhanced efficiency fertilizers (EEFs) are produced and used in the form of N-metabolizing enzymes in the roots or shoots, with nitrate reductase (NR) being the rate-limiting enzyme, which reduces nitrate to nitrite and ultimately to ammonium by nitrite reductase (NiR).The ammonium is then converted into glutamine and glutamate by glutamine synthase (GS) and glutamate synthase (GOGAT), as well as by glutamate dehydrogenase (GDH), which are used for the synthesis of amino acids, proteins, and other N molecules (Hawkesford and Griffiths, 2019;Iqbal et al., 2020b).These molecules are then translocated from source to sink, acting as precursors for nitrogenous compounds and becoming part of the internal N pool.This internal nitrogen pool regulates absorption, translocation, and metabolism, with specific genes encoding these key enzymes serving as an indicator for genotypic responses based on external N availability (Wang et al., 2021).It has been reported that the GS overexpressing can increase NUtE and NUpE in wheat and barley, leading to increased crop yields (Perchlik and Tegeder, 2017;Quan et al., 2017).Research on NR, GS, and GOGAT expression levels in different genotypes of cotton cultivars also found significant differences among different cultivars (Iqbal et al., 2020a).Therefore, understanding different steps involved in N uptake and assimilation, along with enzymes corroborated with specific genes identified as hotspots for NUE, can help identify important features contributing to nitrogen utilization (NUtE) (Hakeem et al., 2012).Studies have shown that root traits play a crucial role in nitrogen uptake (NUpE), with high N-efficiency cultivars exhibiting higher absorption, length, surface area, and volume compared to low N-efficiency cultivars (Khan et al., 2020;Bashir et al., 2023).These traits improve dry matter production capacity under low nitrogen supply; however, the dynamic nature of nitrogen and its loss from soil-plant systems presents challenges for efficient N uptake and management (Li et al., 2020;Tantray et al., 2022).Aeroponics has been perceived as a technology-crammed innovation that serves as a benchmark for potato mini-tuber production research and up-scaling programmes, facilitates N uptake mechanisms study, and enable researchers to use a non-invasive means to examine root and shoot characteristics, making it suitable for year-round potato cultivation, regardless of crop season (Tiwari et al., 2018;Eldridge et al., 2020;Muratore et al., 2021).Up to now, the underlying regulatory mechanism of the PASP-Ca synergist, with particular reference to Nmetabolism, and related changes at enzymatic and molecular levels to induce high increases in NUE in potato plants have not been explored.Therefore, challenging, comprehensive investigation at physio-biochemical, enzymatic, and molecular levels is warranted to fully apprehend the in-depth regulatory mechanism of PASP-Ca within plants or its interaction with biological molecules to improve nitrogen utilization.Based on the importance of the above-mentioned facts, the current research was conducted in aeroponics to get a profound perception into the regulatory mechanism and response of contrasting potato genotypes under PASP-Ca addition at different N supplies by investigating plant growth, N-related parameters, root morphological parameters, photosynthetic gas exchange parameters, and enzymatic parameters coupled with their gene expression profiles (leaves and roots).Thus, providing theoretical and practical aspects of optimizing PASP-Ca in enhancing nitrogen utilization, plant growth, and yields in potatoes, especially at low nitrogen supply.

Plant material and aeroponic cultivation
In this study, four contrasting potato genotypes were selected based on previous screening for biomass and NUE in an aeroponically cultured experiment, including two N-efficient; Feureta (FW) & Qingshu-9 (C17) and two Ninefficient; Chuanyu-56 (C49) & Liangshu-97 (C11) genotypes, and multiplied in vitro from replicated nodal cuttings on MS medium with little modifications in the growth room of the Research and Development Centre of Potato, College of Agronomy, Sichuan Agricultural University, China.After that, morphologically uniform 25-day-old tissue cultured plantlets without roots were hydroponically cultured in basins containing full-strength Hoagland nutrient solution under controlled conditions (at approx.25°C, 60% RH characterized by 16:8 h light-dark cycle) for 20~25 days, to ensure successful acclimatization before transplantation.After pricking out with an average height of 15~20 cm, these micro plants were transplanted in aeroponic boxes at a spacing of 10 cm×15 cm (54 plants per m 2 ), with a plot area of 2.4 m 2 , in the greenhouse at the Crop Research Institute of the Sichuan Academy of Agricultural Sciences, Chengdu, Southwest China (30°66 N, 103°54 E, 500m above sea level).Once the plants were well-established, the lower 2-3 compound leaves were removed and the plants were pulled down to stimulate the growth of roots and stolon's while preventing minitubers initiation in the planting holes.A manual staking system was also installed by tying the plants with thread to guide the upward growth of the stems.

Experimental design
Asplit-split plot design was adopted, with the main plots subjecting to nitrogen treatments, (low, 60 mg/L) and high, 420 mg/L), with a consistent NH4 + to NO3 -ratio (1:9) in each treatment; sub-plots were assigned to N-efficient (FW & C17) and N-inefficient (C49 & C11) potato genotypes, while the sub-sub-plots were set by polyaspartic calcium (PASP-Ca) treatments (CK, 100 & 200 mg/L), respectively (Table 1).All reagents used in nutrient solution preparation were analytically pure (AR) (Table 2).In the early stage of planting, nutrient solutions were periodically sprayed for 30 seconds every 5 minutes, and after rooting, they were adjusted to 30 seconds every 10 minutes to maintain root moisture and aerial turgidity.Nutrient solutions were renewed weekly, with pH and electrical conductivity (E.C.) maintained between 5.5-6.5, and 1.5-2.0dS/m, respectively.After planting for 14 days, foliar application of PASP-Ca treatments was done early in the morning during active plant growth once a week for 14~56 days in total, 6 times, and from 63~90 days with distilled water, 2 times.For control treatment, distilled water was sprayed once a week for 14~ 90 days, in total 8 times.A cardboard border was placed between treatments while spraying to avoid drifting to adjacent treatments.PASP-Ca synergist was provided by Desai Chemical Co., Ltd., China, having a molecular mass of 6934.51Da, a viscosity of 0.062, and its chemical structure as shown in Fig. 1.
During the experiment, appropriate pest control was also carried out.Plants were collected for physio-biochemical, NUE, enzymatic, and gene expression analyses according to set protocols.All traits were measured by pooling five plants from each treatment combination and presented here on a per-plant basis.

Phenotypic and yield traits
Plant phenotypic parameters such as height and Stem diameter, were measured at 35, 52 and 77 days after transplanting (DAP), respectively.For dry matter accumulation (leaf, stem root and total biomass), plant samples were oven dried at 105 °C for 30 mins, and then at 80 °C till constant biomass, were weighted instantly after taking out from the oven.Mini-tuber harvesting commenced around 35-40 DAP, depending on the cultivar, and was done sequentially once every two weeks.There was 5 harvests made over the course of the growing period.For examining yield, 50 plants from each plot were selected, and the number of original seeds taken for each harvest was noted and counted.All micro-tubers weighing more than or equal to 3 g were harvested in the first four harvests, and all microtubers weighing more than or equal to 0.5 g were harvested in the final harvest.The original stock for each harvest was separated into 5 grades: 0.5-1, 1-3, 3-5, 5-10, and >10 g.The number of tubers per plant and yield were calculated as the average of the 50 plants assessed (Wang et al., 2018a;Tiwari et al., 2020c).
Root system architecture and root activity measurement Root morphological parameters (total root length, surface area, root volume, and diameter) were measured using the special image analysis software programme WinRHIZO (Version 2007d, Regent Instrument Inc., Canada) in conjunction with an Epson Perfection V700 scanner (Abenavoli et al., 2016).Cell activity (vitality) in potato roots was analyzed by using the root activity (RA) assay kit (Suzhou Geruisi Biology, Suzhou, China) following the manufacturer's instructions at three different time points: 35, 52, and 77 days after transplanting (DAP), respectively.

Measurement of biochemical indicators
Biochemical indicators were quantified by employing assay kits from Solarbio life sciences (Beijing, China) following the manufacturer's recommended protocols in tubers at harvest stage.The starch content test kit (Solarbio, Cat#BC0700), and reducing sugar content assay kit (Cat#BC0235) were obtained for subsequent spectrophotometric measurements, providing a more comprehensive analysis.

Measurement of N concentration and NUE traits
N concentrations of plant tissues parts (leaf, stem and root) were measured using an automatic kjeldahl apparatus (Kjeltec 8400, FOSS, Hoganas, Sweden) at three different time points: 35, 52, and 77 (DAP), respectively.N-efficiency traits including NUE, NUpE, NUtE, AgNUE, HI and NHI were calculated according to (Jiang et al., 2013) at harvest stage .

Gene expression analysis
The haul-out of total RNA from the leaf and root tissues (100 mg each) at three different time points: 35, 52, and 77 DAP, was done using the RNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands) and reversely transcribed using the Omniscript RT Kit (Qiagen).Quantitative real-time PCR (qPCR) was performed using the SYBR Premix Ex Taq (TaKaRa, Dalian, China) and the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).N assimilation-related gene sequences were obtained from the online sequence database NCBI based on their putative involvement in N-uptake and assimilation in S.tuberosum.Subsequently, gene-specific primers for qRT-PCR were designed using the software Primer Premier 5.The sequences of forward (F) and reverse (R) primers of selected genes were listed in Table S1.The PCR mix was composed of 5 µl SYBR Premix Ex Taq, cDNA corresponding to 30 ng RNA, 0.3 µl of each F and R primer (10 mM), and PCR-grade water up to the final volume of 10 µl.The incubation temperature was: (1) denaturation at a cycle of 95 °C for 3 min; (2) amplification: 40 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; (3) final elongation at 72 °C for 3 min; and (4) melting curve analysis (65-95 °C).All reactions were run as duplicates on 96-well plates.The quantification of N assimilation-related genes was evaluated relative to actin using the 2 −ΔΔCt method (Bashir et al., 2023).

Statistical analysis
The statistical analyses were carried out using R (version 4.0.4)package Agricolae (version 1.3-3) (de Mendiburu and de Mendiburu, 2019).Following validation of the normality and homogeneity assumptions, a two-way ANOVA was performed, and means were separated using Tukey's Honest Significant Difference (HSD) test (p ≤ 0.05).
StepOneTM software v.2.3 was used to analyze qPCR data.Figures were generated in the R environment using the ggplot2 package (Wickham et al., 2016).All data were presented as the mean standard error (SE) of three technical and biological replicates.The correlation between gene expression, morpho-physiological, NUE, and biochemical characteristics were calculated in R using the GeneNT package (version 1.4.1), and the results were visualized using Metscape (version 3.1.3)(Karnovsky et al., 2012).
Yield traits were significantly influenced by N levels, but LN-P100 showed the maximum results as compared to LN-PCK, LN-P200, HN-PCK, HN-P100, and HN-P200 treatments, respectively.A clear genotypic difference was observed for yield attributes, where C17 had highest number of tubers per plant (19%), tuber yield per plant (23%), tuber number per unit area (7%), and tuber yield per unit area (24%), as compared to C49, respectively.Moreover, after the application of PASP-Ca synergist, increase in tubers yield increased at low N levels and then at high N levels in C17 as well as in C11 genotype, indicating their high efficiency and potential at relatively reduced N fertilization.The proportion of 0.5-1 g of original stock of minituber was the highest (27%) for C17 at all treatments, respectively.With the increase in nitrogen level, the proportion of 1-3 g minitubers of all genotypes did not change much.The proportions of 3-5 and 5-10 g potatoes were the highest when the PASP Ca level was LN-P100, which were 24% and 16% for C17 as compared to C49, respectively.There was no significant difference for micro tubers larger than 10 g for both nitrogen levels in conjunction with PASP-Ca for all genotypes (Fig. 2, S1c).
Different PASP-Ca treatments under low and high N supplies differentially influenced the leaf photosynthetic traits of potato genotypes, such as net photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO2 concentration.The results showed that LN-P100 significantly increased photosynthetic rate (12%), stomatal conductance (20%), and transpiration rate (12%), whereas intercellular CO2 concentration was (4%) lower as compared to control and other PASP-Ca treatments.In particular, C17 exhibited the highest photosynthetic rate (10%), stomatal conductance (9%), transpiration rate (10%), and intercellular CO2 concentration (11%), less than C49, which had the lowest values for these three traits but the highest intercellular CO2 concentration at each PASP-Ca level.The high intercellular CO2 concentration levels under high N supply in C49 indicated its lower efficiency in carboxylating the available carbon dioxide (Fig. S2).

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Impacts of PASP-Ca synergist on RSA and RA under different nitrogen levels Root morphology of aeroponically grown potato genotypes were studied under different N levels in conjunction with PASP-Ca treatments.PASP-Ca level (100 mgL _1 ) at low and high nitrogen supplies extensively improved root length, root surface areas, root diameter, and root volume in nitrogen efficient as well as in inefficient genotypes.However, as the PASP-Ca levels were further increased, a decreasing trend in these root traits became evident.At each PASP-Ca level, the percent increase in each root trait was highest in genotype C17, compared to other genotypes, particularly C49.Under LN-P100, root morphological traits like root lengths, root surface areas, root diameter, and root volume were increased by (13%, 19%, 14%), (13%, 2%, 5%), (20%, 9%, 10%), and (22%, 5%, 4%) at 35, 52, and 77 DAP, respectively as compared to other treatments.In comparison with C49, C17 had significantly higher root length (13%, 15%, 11%), root surface area (9%, 4%, 13%), root volume (20%, 10%, 7%), and root diameter (28%, 9%, 3%) followed by C11 at corresponding time points, respectively (Fig. S3).Root activity, being a comprehensive index, reflects root absorption function in potatoes.A decreasing trend in root activity was observed from 35 DPA to 77 DAP; with the lowest values recorded at 77 DAP.Interestinly, the application of PASP-Ca level (100 mgL -1 ), both under low and high nitrogen supplies, extensively improved root activity in nitrogen efficient and inefficient genotypes but decreased with further increase in PASP-Ca levels.Among all treatments, the LN-P100 treatment increased root activity by 20%, 13%, and 9% at 35, 52, and 77 DAP, respectively.Notably, a clear genotypic difference was noted, C17 had significantly highest root activity, 26% at 35 Dap, 16% at 52 Dap, and 14% at 77 DAP, as compared to other genotypes.We concluded from the above analysis that the effect of nitrogen in increasing root activity would gradually decreases with increasing nitrogen fertilization levels, irrespective of PASP-Ca levels and genotypes (Fig. 3).
{Insert Fig. 3. here} Influence of PASP-Ca synergist on starch and reducing sugar contents under different nitrogen levels The upsurge in biochemical traits, including starch and reducing sugar content of aeroponically grown potato tubers with varying nitrogen efficiencies, was observed and showed the same pattern as we incrementally enhanced the PASP-Ca dose from control to 100 mgL _1 under both low and high nitrogen levels.Under LN-P100, a significant rise in these biochemical traits (38%) and (35%) was noted in comparison with other PASP-Ca doses.Among the genotypes, this upsurge was highest in C17 by 53% (starch content) and 45% (reducing sugar content) as compared to C49, respectively (Fig. S6) Influence of PASP-Ca synergist on N accumulation and NUE traits under different nitrogen levels Significant differences (P≤0.05) in leaf, stem, and root nitrogen concentrations were observed for efficient and inefficient potato genotypes under low and high N levels in combination with PASP-Ca treatments.However, results showed that LN-P100 significantly increased nitrogen concentrations in leaf (33%, 37%, 28%), stem (29%, 36%, 30%), and root (29%, 35%, 31%) at all DAPs, in comparison with other PASP-Ca doses at low and high N levels, ensuring that the application of PASP-Ca synergist promotes the accumulation of nitrogen even at low nitrogen levels.
Similarly, a significant difference (P≤0.05) in the leaf, stem, and root nitrogen concentrations was observed for potato genotypes.The increasing trend in N-accumulation was as C17>C11>FW>C49 in leaf, stem, and roots at all DAPs.
As shown in (Fig. S4b) significant differences (< 0.05) were observed in N-efficient and inefficient potato genotypes for NUE traits (NUE, NUpE, NUtE, AgNUE, HI, and NHI) in aeroponics under low and high N levels in conjunction with PASP-Ca treatments.NUpE increased at high N levels as compared to low N; however, in our results after the application of PASP-Ca synergist, it also improved NUpE at low N levels, especially at PASP-Ca doses (100 mg/L).
NUpE was significantly increased by 41% under LN-P100 at low N as compared to high N.Likewise, under LN-P100, AgNUE also improved by 54% as compared to LN-PCK, LN-P200, HN-PCK, HN-P100, and HN-P200 treatments, indicating more N allocation or remobilization towards root under low N concentration.Conversely, NUtE decreased with an increase in nitrogen supply; however, low nitrogen levels improved nitrogen utilization efficiency by 34% after the application of PASP-Ca, particularly at the PASP-Ca dose (100 mg/L).Most importantly, at low and high nitrogen supplies, the PASP-Ca dose (100 mg/L) extensively improved NUE (42%), HI (39%), and NHI (48%), respectively.
Irrespective of the N levels combined with PASP-Ca treatments, NUpE, AgNUE, and NUtE in C17 were 14%, 10%, and 20% higher than other genotypes, especially C11 and FW.Furthermore, NUE was significantly enhanced in Nefficient potato genotypes, C17 (49%) followed by FW (41%), as compared to N-inefficient genotypes.Meanwhile, HI was significantly enhanced in C11 (49%), followed by C17 (35%), while NHI was significantly higher in C17 (56%), as compared to other genotypes.Obviously, the current results proved that C17 endorses being more efficient in obtaining, allocating, and using N for metabolism.

Effects of PASP-Ca synergist in regulating N assimilating enzymatic activities under different nitrogen levels
The dynamic activity of N assimilating enzymes (NR, GS, GOGAT, and GDH) in the shoot and root of N-efficient and inefficient potato genotypes were studied in response to various PASP-Ca treatments under low and high N levels at different growth stages.A clear reduction in N enzymatic activities was observed from 35 to 77 DAP, and the values for these enzymes were greatly reduced at 77 DAP.The peak activity of shoot and root reductase (NR) was observed under LN-P100 treatment, with an increase of (22%, 20%, 18%) and (13%, 18%, 16%) at 35, 52, and 77 DAP, respectively.The genotypic difference for NR activity was obvious, and C17 had significantly higher shoot and root NR activity as compared to C49.Shoot NR activity in C17 increased by 26%, 21%, and 19%, whereas root NR activity increased by 15%, 14%, and 9% at 35, 52, and 77 DAP.The difference in the shoot and root NR activity of C11 at high N level with PASP-Ca doses (100 and 200 mg/L) was similar to that of C17 at low N level combined with PASP-Ca doses (CK and 100 mg/L), indicating the high efficiency of C17 for N metabolism (Fig. 5Sa).
Glutamine synthetase (GS) is one of the main enzymes for amino acid synthesis in nitrogen metabolism.Shoot and root GS activity of potato genotypes decreases from 35 to 77 DAP, and more reduction was noted at 77 DAP.An increasing trend was observed in GS activity for shoot and root at PASP-Ca level (100 mg/L) at low and high nitrogen supplies for all genotypes.A significant increase of 11%, 12%, and 14% in shoot GS activity and 11%, 12%, and 14% in root GS activity was observed under LN-P100 treatment at 35, 52, and 77 DAP, respectively.At each N level concomitant with LN-P100 treatment, by comparison, shoot and root GS activity was significantly increased in C17 (6%, 9%, 7%) and (10%, 15%, 12%), relative to C49 at all DAPs, respectively (Fig. 5Sb).

Effects of PASP-Ca synergist on the transcript levels of key enzymes involved in N Metabolism
Expression profiles of nitrate reductase gene StNR involved in N Metabolism in response to PASP-Ca synergist The transcript levels of key enzyme genes involved in N metabolism-encoding StNR (nitrate reductase), StGS (glutamine synthetase), StGDH (glutamate dehydrogenase), and StGOGAT (glutamate synthase) were analyzed in the leaves and roots of N-efficient and N-inefficient potato genotypes, in response to various PASP-Ca treatments (CK, 100, and 200 mg/L) under low and high N levels at three different time points: 35, 52, and 77 DAP employing RT-qPCR.The expression of StNR generally exhibited an upward trend from 35 to 77 DAP after the application of PASP-Ca synergist under low and high N levels.Under LN-P100 treatment, substantially higher expression of StNR was noted in both leaves and roots of C17 followed by FW and C11, relative to other treatments, particularly evident at 77 DAP, indicating that PASP-Ca application could facilitate the expression of StNR.It might be that the application of PASP-Ca synergist surged up the assimilation of nitrate and catalyzed the conversion of nitrate to nitrite (Fig. S7).
Relative expression analysis of nitrate transporter genes StNRTs involved in N Metabolism in response to PASP-Ca synergist.
StNRTs are nitrate transporters that aid in the absorption and transportation of nitrate N, sense external N signals, and promote plant growth.StNRTs include StNRT2.5 and StNRT2.7.In comparison to other treatments, the expression level of StNRT2.5 in leaves under LN-P100 treatment peaked at 35 DAP in C17, followed by FW and C11, showing a trend of increasing first and then decreasing from 35 to 77 DAP.On the other hand, StNRT2.5 expression in roots generally showed an upward trend under LN-P100 relative to other treatments, peaking at 52 DAP after initially decreasing and then increasing from 35 to 77 DAP.The most pronounced downregulation in the expression of StNRT2.5 was noted in C49, while the minimum downregulation in C17 among genotypes (Fig. S8).The relative expression for StNRT2.7 exhibited a linearly declining pattern from 35 to 77 DAP, while LN-P100 showed the maximum results as compared to other treatments at low N levels, but marginally increased at high N levels, albeit not significantly, showing the same trend in leaves and roots.StNRT2.7 was relatively abundant in the first half of the growth stage and relatively lowest at 77 DAP.A distinct genotypic difference was noted, C17 had significantly higher expression of StNRT2.7,followed by C11 across all DAPs in leaves and roots.From the overall analysis, it was found that the relative expression levels of these two genes in roots were higher than those in leaves, which could be attributed to the roots capacity to initially and directly perceive external N signals, hence regulating N absorption and transport (Fig. S9).

Relatedness of glutamine synthetase genes StGSs in response to PASP-Ca synergist under different nitrogen levels
In order to facilitate the conversion of inorganic nitrogen into organic nitrogen, ammonia absorption requires the StGS genes, mainly including StGS1, StGS1-2, StGS1-3, and StGS2.The most significant upsurge in transcript levels of StGS1 was noticed with an increasing PASP-Ca level from control to 100 mg/L at low N supplies in the tissues (leaves and roots) at all growth stages, with an initial increase at 35 DAP, reaching a peak at 52 DAP, followed by a decrease at 77 DAP, while maximum escalation was observed in C17 and a minimum upsurge in C49 among genotypes (Fig. S10) The relative expression for StGS1-2 exhibited a linearly decreasing trend from 35 to 77 DAP, albeit marginally greater at 35 DAP, while LN-P100 showed the maximum results as compared to other treatments at low N levels, showing the same trend in leaves and roots.Among all genotypes, the maximum expression of StGS1-2 was pronounced in C17, while the lowest expression was found in C49 (Fig. S11).StGS1-3 showed significant differences in the expression sites, roots and leaves.The expression of StGS1-3 generally exhibited an upward trend from 35 to 77 DAP after the application of PASP-Ca synergist under low and high N levels.Under LN-P100 treatment, maximum and equally significant expression of StGS1-3 was noted for C17, followed by C11, relative to other treatments; however, this was evident at 77 DAP. (Fig. S12).The overall expression of StGS2 in roots and leaves increased initially and then gradually decreased as the growth stage progressed and continued to show a decreasing trend in the later stage, with maximum expression at 52 DAP and most obvious in roots.In comparison to LN-PCK, LN-P200, HN-P100, and HN-P200, LN-P100 was found to be the most effective at low and high N levels.In terms of StGS2 expression upregulation, genotype C17 showed the maximum escalation, whereas genotype C49 exhibited the least (Fig. S13).While LN-P100 showed the maximum results as compared to LN-PCK, LN-P200, HN-PCK, HN-P100, and HN-P200 treatments.While a clear genotypic difference was noted, C17 had significantly higher expression of Fd-GOGAT, followed by C11 across all DAPs.In roots, contrastingly, the expression of StFd-GOGAT was observed more abundant in the second half of the mid-growth stages form 52-77 DAP.Under LN-P100 treatment, substantially higher expression of StFd-GOGAT was noted in C17 (3.82-4.05folds) followed by C11 (3.33-3.86folds) and FW (3.22-3.57folds), relative to other treatments (Fig. 3).The transcript level of StNADH-GOGAT was highly significant with growth stages; where it first decreased and then subsequently increased at high N levels compared to low N.However, our results demonstrated that after applying PASP-Ca synergist, it also improved its expression at low N levels, especially at doses of 100 mg/L, and then decreased while it was abundant in the second half of the mid-growth stage at 52 DAP in leaves.In roots, the expression trend at each stage was consistent with that in leaves.At each N-level, concurrent with LN-P100 treatment, the upsurge in relative expression of StNADH-GOGAT was C17>C11>FW>C49 in both leaves and roots at all DAPs (Fig. 4 StGDH is a key enzyme gene that catalyzes the glutamate deamination into α-ketoglutarate and participates in the TCA cycle, thereby affecting N-assimilation.The most significant upsurge in transcript levels of StGDH was noticed with an increasing PASP-Ca level from control to 100 mg/L at low N supplies in the tissues (leaves and roots) of C17 and a minimum up-regulation in C49 among genotypes at all DAPs.The expression level at 35 DAP was relatively low, while at 52 DAP and 77 DAP, it first increased and then decreased; however, the escalation was observed to be maximum at 52 DAP in leaves (10.8 folds) and roots (3.61 folds).The results demonstrated that using the PASP-Ca synergist dose (100 mg/L) could promote the expression of StGDH even at low N levels (Fig. S14).

Correlations analysis for key traits mining to enhance NUE via PASP-Ca synergist
To better understand the key traits contributing to NUE, NUpE, and NUtE, a correlation network of various morphophysiological, root-related, NUE-related, enzymatic, biochemical, and genetic traits was constructed based on correlation coefficients.The results showed that 49 nodes (traits) were connected to 1176 edges in the correlation network.Among the direct correlations, NUE emerged as the linchpin, exhibiting the strongest positive correlations with 43 traits, including NUpE and NUtE efficiencies, driving improved NUE with PASP-Ca addition.Intriguingly, NUpE had the strongest positive correlations with 24 traits, medium-strong correlations with two traits, low correlations with four traits, and a strong negative correlation with only one trait.The N-assimilating enzymes were strongly associated with NUpE and may be target traits for augmenting NUE.For NUtE, a total of 22 traits showed a positive correlation.Strong positive correlations were found with 12 traits, medium-strong correlations with 14 traits, low correlations with 4 traits, and a medium negative correlation with only one trait.Notably, the root morphological traits (root dry weight, RSA, RL, RA, and RV) were strongly positive correlated with NUtE and with each other under low N supply, which may be associated with the nitrogen metabolism due to an increase in transcript levels encoding various genes, solidifying their importance as key contributors in enhancing nitrogen utilization (Fig. 6).{Insert Fig. 6. here}

PASP-Ca's influence on Growth and biomass dynamics
Polyaspartic acid-calcium (PASP-Ca) is an eco-friendly amino acid polymer complex predominantly used as a novel synergist for improving nitrogen utilization efficiency (NUE) and productivity of agricultural crops.The physiomorphological characteristics observed in this study shed light on the significant impact of PASP-Ca on potato plant growth and biomass production.The bell-shaped growth curve observed for various traits like plant height, stem diameter, leaf, stem, and root dry matter accumulation at different days after planting (DAP) reflects the dynamic nature of plant development (Fig. S1a,b).Interestingly, the application of PASP-Ca synergist, particularly LN-P100, led to remarkable improvements in these traits, with a more pronounced effect on N-efficient genotypes (C17 and FW).These findings underline the potential of PASP-Ca to enhance crop growth and biomass accumulation, even under limited nitrogen availability.These results agree with the promoting effect of PASP in maize (Wang et al., 2018b) suggesting that the synergist has the potential to enhance the growth and biomass production of potato plants, even at reduced nitrogen fertilization levels PASP-Ca's influence on yield and photosynthesis Yield parameters, a critical determinant of agricultural productivity, exhibited substantial improvements with the addition of PASP-Ca, especially at low nitrogen level, realize the synchronization of nutrient supply and potato nutrient absorption in time and the matching of fertilizer amount.The findings indicated that PASP-Ca significantly enhance tuber yield, tuber number, and tuber size, which are pivotal factors for economic returns, particularly in genotype C17, highlights the importance of selecting suitable potato cultivars for efficient nitrogen utilization.The current results are consistent with the previous studies performed in rice (Deng et al., 2014;MeiX, 2019;Yan et al., 2022), where the application of PASP-urea increase the grain yield.Notably, the proportion of larger-sized minitubers increased with PASP-Ca treatment, suggesting its role in enhancing the yield of marketable tubers, a factor of paramount importance to farmers and the potato processing industry (Fig. 2, S1c).Similarly, photosynthesis is very sensitive to changes in N availability (Xu et al., 2015), because 57% of the N in the leaves is located in the chloroplasts and is used for the synthesis of photosynthetic components and related enzymes.The photosynthetic capacity is positively correlated with leaf N content (Bashir et al., 2023), and consistently, a positive correlation was noted between N concentration and photosynthetic activity.Improved photosynthetic traits were observed in plants treated with LN-P100.Enhanced photosynthetic rates, stomatal conductance, and transpiration rates, coupled with lower intercellular CO2 concentration, indicate improved photosynthetic efficiency.Notably, lower intercellular CO2 concentration under LN-P100 treatment indicates more efficient carbon dioxide utilization, which is essential for enhanced photosynthesis and crop growth.These changes may result from the enhanced nutrient availability facilitated by PASP-Ca, leading to greater carbon assimilation and subsequent growth (Fig. S2).The observed differences among genotypes suggest that some potato varieties may be more responsive to PASP-Ca treatment in terms of photosynthetic performance (Theerawitaya et al., 2023).Increasing trends were observed for the photosynthetic activity of C17, as reported in poplar species by (Luo et al., 2015).In contrast, the photosynthetic activity of C49 decreased, which may be associated with an inhibited photosystem, because many genes involved in photosystem were downregulated under high-N conditions (Luo et al., 2015).The reduction in the overall photosynthetic efficiency levels of potato genotypes under high N concentrations at control and HN-P200 may results from the decrease in carboxylation efficiency as the intercellular CO2 concentration increases, matched with the finding of (Huang et al., 2004;Du et al., 2020) in rice and sunflower.Thus, N is important for photosynthesis, which is the basis for increasing crop growth, productivity, and NUE.

Mechanism of PASP-Ca synergist on RSA and nitrogen assimilation
One of the key findings of this study was the substantial improvement in root system architecture (RSA) parameters, encompassing root length, surface area, diameter, and volume, in response to PASP-Ca treatment, particularly LN-P100, with a more pronounced effect on N-efficient genotypes, thereby augmenting their nutrient acquisition capacity.
High RSA is paramount for efficient nutrient uptake, especially under low nitrogen conditions (Fig. S3).The roots of C17 were more sensitive to low-N concentration than other genotypes indicating that high N-efficiency cultivars exhibit superior root traits underscoring that if the root system is developed, thick and long, the nutrient absorption area will be large and the root system will be vigorous, thereby expand the effective absorption space of the root system, ultimately improving the absorption efficiency of nutrients as observed in rapeseed and cotton (Iqbal et al., 2019;Qin et al., 2019).The most probable mechanism may be that PASP-Ca being polypeptide in nature play an important role as a signaling compound (Adelnia et al., 2021).Specific receptors on cell membrane interact with signal transduction polypeptides, changing cell membrane permeability and promoting biosynthesis of endogenous plant hormones.The mechanism of PASP-Ca action on RSA and nitrogen assimilation could be related to its effects on cell wall metabolism and auxin signaling.PASP-Ca increased the expression of XTH31 gene involved in cell wall modification, respectively.XTH31 encodes a xyloglucan endotransglucosylase/hydrolase that can loosen the cell wall and facilitate cell expansion (Zhang et al., 2022).The up-regulation of this gene could promote root elongation and shoot growth by increasing cell wall extensibility and energy supply.Moreover, PASP-Ca enhanced the expression of AIR12 and SFC, two genes involved in auxin biosynthesis and transport, respectively.AIR12 encodes an auxinresponsive protein that can modulate auxin homeostasis and signaling (Preger et al., 2009).SFC encodes a GTPaseactivating protein that can regulate auxin polar transport by affecting the localization of PIN proteins (Sieburth et al., 2006).The up-regulation of these genes could stimulate root development and lateral branching by increasing auxin synthesis and distribution and lead to morphological, physiological and biochemical changes of plant roots.Root activity, as a comprehensive indicator of nutrient absorption, exhibited an increasing trend under PASP-Ca treatment, further highlighting its positive impact on nutrient uptake.Moreover, the increase in root activity, particularly in nitrogen-efficient genotypes, suggests that PASP-Ca treatment augmented the root's capacity to efficiently absorb and assimilate nitrogen (Fig. 3), which was consistent with previous research results (Jiang et al., 2013;Zhang et al., 2013;Hu et al., 2019;Zhang et al., 2021).

PASP-Ca synergist enhances carbohydrate metabolism and nitrogen accumulation, unveiling genetic variability in nitgrogen utilization and key enzymatic pathways in potato crop
Carbohydrate metabolism represented by starch and reducing sugar content in potato tubers, exhibited significant improvements following PASP-Ca treatment, especially LN-P100 in N-efficient genotypes (C17) (Fig. S6).Elevated starch content is particularly noteworthy, given its implications for tuber quality and marketability (Li et al., 2018).
These results suggest that PASP-Ca can influence carbohydrate metabolism, potentially through its impact on photosynthesis and upregulating the expression of BFRUCT4 involved in carbohydrate translocation to tubers, matched with the findings of (Xiong and Ma, 2022) in brassica under different N supplies, respectively.
Nitrogen accumulation in different pant parts, including leaves, stems, and roots, exhibited significant improvements under PASP-Ca treatment (Fig. S4a).Notably, PASP-Ca's capacity to enhance nitrogen accumulation, even at low nitrogen levels, highlights its role in promoting nitrogen use efficiency (Hu et al., 2019).The differential responses among genotypes underscore the genetic variability in nitrogen uptake and utilization, with N-efficient genotypes displaying superior performance as also reported in rice (Bashir et al., 2023).Increasing the NUE is important to maintain a high productivity level with a comparatively low N supply (Iqbal et al., 2020b).Improvements in NUE traits, including NUpE, NUtE, AgNUE, HI, and NHI, were evident under PASP-Ca treatment (Fig. S4b).The substantial increase in NUE, especially in N-efficient genotypes like C17 and FW, indicates that PASP-Ca has the potential to enhance nitrogen utilization in potato crops.NUpE, which reflects nitrogen uptake efficiency, displayed notable improvements, even at low nitrogen levels, further highlighting PASP-Ca's role in optimizing nutrient acquisition.The increase in AgNUE suggested more efficient allocation or remobilization of nitrogen resources, favoring root growth (Tiwari et al., 2022).Additionally, the improvement in NUtE at low nitrogen levels indicates that PASP-Ca can facilitate nitrogen utilization and assimilation processes, contributing to improved crop performance as observed in rice and maize (MeiX, 2019;Yang et al., 2019).Changes in key enzymatic processes involved in nitrogen metabolism further supported the improvement in NUE (Khan et al., 2020).In both the shoots and the roots, PASP-Ca treatment increased the activity of nitrate reductase (NR) (Fig. 5Sa), glutamine synthase (GS) (Fig. 5Sb), glutamate synthase (GOGAT) (Fig. 5Sc), and glutamate dehydrogenase (GDH) (Fig. 5Sd) (Hakeem et al., 2012;Iqbal et al., 2020b).These enzymes play pivotal roles in nitrogen assimilation and transport, and their upregulation suggests a more efficient use of available nitrogen resources (The et al., 2021).It was speculated that PASP-Ca might influence the enzymatic machinery governing nitrogen metabolism, ultimately contributing to improving NUE in potato crops.
In addition, large genotype-dependent differences were noted in the key enzymes regulating N metabolism, suggesting genetic variations in their nitrogen metabolism pathways (Sahay et al., 2021;Bashir et al., 2023) Interestingly, enzymatic activities in C17 were the highest among the studied genotypes, indicating its greater potential for N metabolism.The variations in N-based enzymatic activities among the genotypes may be associated with differences in regulation of N transporters or N fluxes in the roots (Hsieh et al., 2018).This aligns with previous research indicating that such differences in transporter activity can significantly impact nitrogen acquisition (Kumari et al., 2021;Yang et al., 2021b).Thus, in C17, the low-affinity transporters may be more active in the roots and shoots, which might lead to the high N uptake and assimilation (Gupta et al., 2012).In addition, the high-N metabolism in C17 might result from the constant conversion of nitrate to nitrite and then to ammonia and amino acids, even at low N concentrations (Vijayalakshmi et al., 2015).Additionally, the high N efficiency of C17 may result from a well-coordinated system of N uptake (Yang et al., 2021a) and may be the base of the high N use efficiency in C17, which was explained by their high enzymatic activities.
Deciphering the molecular mechanisms and trait correlations in enhancing NUE in potato genotypes through PASP-Ca synergist enzymes, which could serve as promising targets for enhancing overall NUE (The et al., 2021).In the context of NUtE, root morphological traits, including root dry weight, root surface area (RSA), root length (RL), root activity (RA), and root volume (RV), were highly correlated with NUtE.These root traits were also interrelated, especially under low N conditions, possibly due to their role in influencing nitrogen metabolism, as suggested by the upregulation of genes associated with nitrogen metabolism.Conclusively, root morphological traits may be used as target traits for increasing NUE (Fig. 6).The root is considered the key to a new revolution in agriculture in which crops are developed that can produce more with low fertilizer inputs or NUEs (Muratore et al., 2021).The influence of N on root development is still not clearly understood.Modifying the root architecture is a good strategy for developing crops that can efficiently uptake nutrients even under low-availability conditions (De Pessemier et al., 2013).Recently, natural variations in Arabidopsis accessions were explored to define ideotypes for increasing yield (Li et al., 2017).
In a similar manner, we can learn from the genetic and environmental regulation of root growth and development in potato genotypes, especially C17, which produced a highly branched and long root system at low N concentration, in aeroponics can form an interesting ideotype for efficient N uptake and assimilation suggesting that there may be a synergistic interaction between these traits in promoting NUE.{Insert Fig. 7. here}

Conclusion
In our study, we investigated the impacts of PASP-Ca synergist on potato crop grown aeroponically under varying nitrogen supplies.The crux of our findings are summarised in a schematic diagram (Fig. 7) that illustrates the regulatory mechanism involved.PASP-Ca treatment significantly enhanced plant growth, augmented biomass accumulation, improved root system architecture, and increase in yield parameters particularly in N-efficient genotypes.Additionally, positive effects of PASP-Ca were ascribed by its ability to improve photosynthesis, upregulation of key genes increasing the activity of nitrogen-metabolising enzymes in both root and shoot, with a stronger effect in roots, highlighting their importance in NUE, especially under low nitrogen conditions.Our study identified C17 and C49 as the most distinct N-efficient and inefficient genotypes based on various traits.Notably, PASP-Ca at 100 mg/L was the most effective dose for enhancing NUE under aeroponics conditions.Overall, our study signifies a pioneering endeavor in highlighting PASP-Ca synergist as an environmentally sustainable and economically viable strategy to optimise NUE and nitrogen fertiliser inputs, all while upholding high crop yields.
Further research at the proteomic level is warranted to fully understand the molecular mechanisms underlying the PASP-Ca-mediated improvements in NUE.

Figure legends
StGOGATs in response to PASP-Ca synergist under different nitrogen levels StGOGATs are rate-limiting enzyme genes in the N-assimilation GS/GOGAT pathway and are divided into StFd-GOGAT with ferredoxin Fd as the electron donor and StNADH-GOGAT with NADH as the electron donor.The expression trend for StFd-GOGAT in leaves was found relatively consistent, albeit marginally greater at 35 DAP.
) {Insert Fig.4.here} {Insert Fig.5.here} Relative expression analysis of glutamate dehydrogenase gene StGDH in response to PASP-Ca synergist under different nitrogen levels

Fig. 4 .
Fig. 4. Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of the glutamate synthase gene (StNADH-GOGAT) in (A) leaf and (B) root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. 5 .
Fig. 5. Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of the glutamate synthase gene (StFd-GOGAT) in (A) Leaf, and (B) Root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. 6 .
Fig.6.Pearson correlation network reveals the regulatory mechanism of NUE after the application of PASP-Ca synergist in potato crops.Relationships between morphological (purple), physiological (pink), root (light green), NUE-related (yellow), enzymatic (green), biochemical (magenta), and genetic (orange) traits with NUE, NUpE, and NUtE (yellow).Nodes represent the traits, and edges (red, positive; blue, negative) represent the correlations.The thickness of the edges represents the strength of the correlation coefficient for each pair, where strong positive correlations were highlighted by red (r = 0.99) and strong negative correlations by green (r = -0.95 to -0.98).PH, plant

Fig. 7 .
Fig.7.A schematic diagram highlights the enhanced regulatory mechanism of PASP-Ca synergist on potato crop grown in aeroponics under N supply.PASP-Ca treatment significantly improved various physiological and morphological traits in potato plant, including increased plant growth, biomass accumulation, root system architecture, tuber yield.PASP-Ca also showed positive impact on photosynthesis and nitrogen metabolism.Furthermore, the underlying molecular mechanism revealed the upregulation of key genes (StNADH-GOGAT, StFd-GOGAT,StGDH,   StGS1, StGS2, StNR, StNRT2.5, and StNRT2.7)associated with nitrogen metabolism and increased activity of essential nitrogen metabolism enzymes in both shoots and roots.

Fig. S5a .
Fig. S5a.Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on (A) Shoot NR activity (U/g mass) and (B) Root NR activity (U/g mass) in contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. S6 .
Fig. S6.Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on (A) Reducing sugar content (µg/g mass) and (B) Starch content (mg/g mass) in contrasting potato genotypes under low and high nitrogen supplies in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. S7 .
Fig. S7.Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of the nitrate reductase gene (StNR) in (A) leaf and (B) root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. S8 .
Fig. S8.Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of the nitrate transporter gene (StNRT2.5) in (A) leaf, and (B) root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. S10 .
Fig. S10.Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of glutamine synthase gene (StGS1) in (A) leaf and (B) root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. S11 .
Fig. S11.Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of the glutamine synthase gene (StGS1-2) in (A) leaf, and (B) root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. S12 .
Fig. S12.Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of the glutamine synthase gene (StGS1-3) in (A) Leaf, and (B) Root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment..

Fig. S13 .
Fig. S13.Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of glutamine synthase gene (StGS2) in (A) leaf and (B) root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. S14 .
Fig. S14.Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of the glutamine synthase gene (StGDH) in (A) leaf, and (B) root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. 4 .
Fig. 4. Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of the glutamate synthase gene (StNADH-GOGAT) in (A) leaf and (B) root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. 5 .
Fig. 5. Effects of PASP-Ca synergist levels (CK, 100, & 200 mg/L) on relative gene expression of the glutamate synthase gene (StFd-GOGAT) in (A) Leaf, and (B) Root of contrasting potato genotypes under low and high nitrogen supplies at 35, 52, and 77 days after transplanting (DAP) in aeroponics.Data represent means ± standard deviations (n = 3); bars labeled with different letters indicate significant differences (p < 0.05) between genotypes within each treatment.

Fig. 6 .
Fig.6.Pearson correlation network reveals the regulatory mechanism of NUE after the application of PASP-Ca synergist in potato crops.Relationships between morphological (purple), physiological (pink), root (light green), NUE-related (yellow), enzymatic (green), biochemical (magenta), and genetic (orange) traits with NUE, NUpE, and NUtE (yellow).Nodes represent the traits, and edges (red, positive; blue, negative) represent the correlations.The thickness of the edges represents the strength of the correlation coefficient for each pair, where strong positive correlations were highlighted by red (r = 0.99) and strong negative correlations by green (r = -0.95 to -0.98).PH, plant

Table legends Table 1 .
Details of plant material & experimental treatments.

Table 2 .
Chemical composition of modified Hoagland nutrient solutions used for the treatments.

Table S1 .
Primer sequences used for quantitative real-time PCR

Table 1 .
Details of plant material & experimental treatments.

Table 2 .
Chemical composition of modified Hoagland nutrient solutions used for the treatments.