Expression Dynamics of lpa1 Gene and Accumulation Pattern of Phytate in Maize Genotypes Possessing opaque2 and crtRB1 Genes at Different Stages of Kernel Development

Phytic acid (PA) acts as a storehouse for the majority of the mineral phosphorous (P) in maize; ~80% of the total P stored as phytate P is not available to monogastric animals and thereby causes eutrophication. In addition, phytic acid chelates positively charged minerals making them unavailable in the diet. The mutant lpa1-1 allele reduces PA more than the wild-type LPA1 allele. Further, mutant gene opaque2 (o2) enhances lysine and tryptophan and crtRB1 enhances provitamin-A (proA) more than wild-type O2 and CRTRB1 alleles, respectively. So far, the expression pattern of the mutant lpa1-1 allele has not been analysed in maize genotypes rich in lysine, tryptophan and proA. Here, we analysed the expression pattern of wild and mutant alleles of LPA1, O2 and CRTRB1 genes in inbreds with (i) mutant lpa1-1, o2 and crtRB1 alleles, (ii) wild-type LPA1 allele and mutant o2 and crtRB1 alleles and (iii) wild-type LPA1, O2 and CRTRB1 alleles at 15, 30 and 45 days after pollination (DAP). The average reduction of PA/total phosphorous (TP) in lpa1-1 mutant inbreds was 29.30% over wild-type LPA1 allele. The o2 and crtRB1-based inbreds possessed ~two-fold higher amounts of lysine and tryptophan, and four-fold higher amounts of proA compared to wild-type alleles. The transcript levels of lpa1-1, o2 and crtRB1 genes in lpa1-1-based inbreds were significantly lower than their wild-type versions across kernel development. The lpa1-1, o2 and crtRB1 genes reached their highest peak at 15 DAP. The correlation of transcript levels of lpa1-1 was positive for PA/TP (r = 0.980), whereas it was negative with inorganic phosphorous (iP) (r = −0.950). The o2 and crtRB1 transcripts showed negative correlations with lysine (r = −0.887) and tryptophan (r = −0.893), and proA (r = −0.940), respectively. This is the first comprehensive study on lpa1-1 expression in the maize inbreds during different kernel development stages. The information generated here offers great potential for comprehending the dynamics of phytic acid regulation in maize.


Introduction
Mineral deficiencies cause severe health concerns including stunted development, prenatal problems, intellectual disabilities and an increased risk of morbidity and death in humans [1]. These minerals are essential for healthy cell development and signalling [2,3]. Population growth and natural disasters warrant demand for nutrient-rich foods which escalated after the COVID-19 pandemic [4]. Addressing the problem of malnutrition through biofortified staple food is a more affordable and sustainable option than food fortification and medical supplementation [5,6]. Phytic acid (PA), also known as myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the main phosphorous (P) storage molecule in seeds [7]. The negatively charged PA chelates positively charged minerals, making them inaccessible to the human gut [8,9]. Phosphorous is accumulated in growing seeds in excess of what is required for normal cellular function during germination. Monogastric animals are unable to digest PA, which is passed through excretion leading to environmental pollution [10,11]. The rise in phosphorous levels in water bodies due to release of PA from animals such as poultry, results in eutrophication or excessive algae growth [12,13]. Therefore, reduction of PA not only improves the nutritional quality of food, but also alleviates malnutrition in addition to reducing the environmental worries [10,14].
Maize (Zea mays ssp. mays) possesses considerable significance as feed, food and raw material in a variety of industrial purposes and is at the centre of a global initiative to address micronutrient deficiency in underdeveloped nations through biofortification [15]. In order to increase intake of high-quality proteins, quality protein maize (QPM) cultivars are nutritionally enhanced. High lysine, tryptophan and provitamin-A and less antinutritional factors, make these maize genotypes nutritionally superior. As compared to other cereals, maize grains have a higher content of PA, which significantly lowers the bioavailability of minerals [14,16]. Although wide genetic variation for mineral content in maize germplasms have been recorded, polygenic nature and strong environmental influence have limited the development of mineral-rich cultivars [16,17]. Several low phytic acid (lpa) mutants viz. lpa1, lpa2, lpa3, lpa1-7 and lpa241 have been reported in maize [18][19][20][21]. Of these, lpa1-1 mutation with a modification in the trans-membrane transporter protein reduces PA by 55-65% and is not associated with harmful effects on seed germination or seedling vigour [19,[22][23][24][25]. Ragi et al. [25] evaluated the lpa1-1-based maize genotypes at multiple locations and observed~35% reduction in phytic acid in the mutant genotypes. They also reported similar agronomic performance of the lpa1-1-based mutant genotypes as compared to the wild-type genotypes.
Breeding efforts at ICAR-Indian Agricultural Research Institute (IARI), New Delhi, have produced a number of sub-tropically adapted lpa1-1-based maize inbreds through marker-assisted selection (MAS) [24,25]. Further, traditional maize contains unbalanced protein which is poor in essential amino acids (lysine and tryptophan) and provitamin-A (proA) [26]. Mutant opaque2 (o2) and crtRB1 genes enhance the lysine, tryptophan and proA in maize [27]. Though significant progress has been made to develop sub-tropically adapted nutritionally rich maize genotypes, no information is available on the expression pattern of lpa1-1 gene at different stages of kernel development. Further, studying the expression of o2 and crtRB1 in lpa1-1-based genotypes would provide an understanding of the combined effects of these genes on different nutritional quality parameters at different kernel development stages. Furthermore, the transcript expression study would shed light on the intricate regulation of these genes in the biosynthesis pathways, regulate micronutrient bioavailability, and help breeders to determine the precise stage of maize kernel harvest. Early stages of maize are the green forms that we can utilise as a green cob (green ears) which is very popular in Asia, whereas mature stages of maize can be utilised in the form of flour, chapattis and porridges. Hence, we targeted 15, 30 and 45 days after pollination (DAP) for the experiment. With this experiment, we can detect the precise stage of harvest where people can utilise the most nutrients available. The present study was therefore undertaken to analyse the (i) accumulation of PA, inorganic phosphorous (iP) and PA/total phosphorous (TP), (ii) expression pattern of the lpa1-1, o2 and crtRB1 genes, and (iii) correlation among the transcript levels and nutrient accumulation at different stages of kernel development.

Genetic Variation for Nutritional Quality Traits
The results of ANOVA revealed significant differences (p < 0.01) among the genotypes and kernel developmental stages for PA, iP, TP, PA/TP, lysine, tryptophan and proA  (Table 1). The interaction of genotype (G) × days after pollination (DAP) also showed significant (p < 0.01) differences for all the nutritional traits ( Table 1). The contribution of genotypes for the total variation was the highest (range: 54.67 to 80.16%) followed by DAP (range: 11.72 to 64.58%) and G × DAP (range: 0.30 to 12.10%) across the nutritional quality traits. The PA, iP and PA/TP varied from 1.08 to 2.84 mg/g, 0.22 to 1.12 mg/g and 50.30 to 92.70%, respectively (Table S2). Across genotypes, lysine, tryptophan and proA ranged from 0.149 to 0.564%, 0.035 to 0.161% and 1.61 to17.26 µg/g, respectively (Table S3).

PA and Associated Traits during Different Stages of Kernel Development
The PA among the lpa-1-1-based inbreds ranged from 1.08 to 1.87 mg/g across 15, 30 and 45 DAP, whereas it varied from 2.10 to 2.84 mg/g among the wild-type inbreds (Table S2; Figure 1a). An average reduction of PA among lpa1-1-based inbreds was 41.68% as compared to wild-type inbreds. Likewise, the iP among lpa1-1-based inbreds varied from 0.91 to 1.12 mg/g with the mean of 1.0 mg/g (Table S2; Figure 1b). However, the mean iP among the wild-type inbreds was 0.50 mg/g with a range of 0.22 to 0.90 mg/g across 15, 30, and 45 DAP. Among the lpa1-1-based inbreds, nearly two-fold increment in iP content was observed over the wild-type inbreds. Mean PA content of lpa1-1-based inbreds ranged from 1.13 mg/g (15 DAP) and 1.77 mg/g (45 DAP). Similar patterns of PA were observed for wild-type inbreds as well. Among the lpa1-1-based inbreds, highest mean iP was observed at 15 DAP (1.03 mg/g) and lowest iP at 45 DAP (0.98 mg/g). In contrast, significant difference of iP was observed among wild-type inbreds with highest iP at 15 DAP and the lowest iP at 45-DAP. The range of PA/TP ratio varied from 50.30 to 66.00% with a mean of 58.73% among lpa1-1-based inbreds (Table S2; Figure 1c). However, the PA/TP ratio among the wild-type (LPA1-1/LPA1-1) inbreds varied from 70.60 to 92.70% with a mean of 83.00%. The mean PA/TP ratio among lpa1-1-based inbreds showed the lowest at 15 DAP (52.60%) and highest at 45-DAP (64.40%). Across the wild-type inbreds, the increasing trend was observed in PA/TP ratio with lowest ratio at 15 DAP (74%) and highest ratio at 45 DAP (91%). The average reduction of PA/TP in lpa1-1 introgressed inbreds was 29.30% as compared to their wild-type versions. The lpa1-1-based inbreds (PMI-PV5-lpa1, PMI-PV6-lpa1, PMI-PV7-lpa1 and PMI-PV8-lpa1) revealed reduction in mean PA/TP ratio of 23.40%, 26.66%, 23.68% and 24.40% over their respective original versions, viz: PMI-PV5, PMI-PV6, PMI-PV7 and PMI-PV8 (Figure 1c).

Variation in Expression of Genes
ANOVA revealed that transcript levels of lpa-1-1, o2 and crtRB1 genes revealed significant differences (p < 0.01) among the genotypes as well as among the different stages of kernel development ( Table 2). The expression level of lpa1-1 transcripts (2 −∆C T ) varied from 0.064 to 2.121 across the genotypes, while it was 0.011 to 0.821 and 0.010 to 0.387 for o2 and crtRB1 genes, respectively (Table 3).

Expression Pattern of o2 Gene during Kernel Development Stages
The lpa1-1 inbreds with mutant o2 allele and HKI1105 possessing wild-type O2 allele showed differences in transcript levels of o2 gene during different stages of kernel development ( Figure 3b). The highest mean expression of o2 gene was observed at 15 DAP (0.067) followed by 30 DAP (0.028), while the lowest expression level was detected at 45 DAP (0.017) across the lpa1-1-based inbreds (Figure 3b). The control inbred HKI1105 showed a similar pattern for o2, with highest expression at 15 DAP and lowest expression at 45 DAP. Among the lpa1-1-based improved lines, the highest expression level of o2 was observed for PMI-PV6-lpa1-A (0.079) at 15 DAP, while the lowest expression level was found for PMI-PV5-lpa1-B (0.012) at 45 DAP. The highest and lowest transcript levels of O2 in HKI1105 were 0.821 and 0.205, respectively. Across the kernel development stages, lpa1-1-based improved lines recorded significantly lower levels of o2 expression (13.07fold lower transcripts) over the wild-type (O2) in control inbred (HKI1105). The inbreds, viz. PMI-PV5, PMI-PV6, PMI-PV7 and PMI-PV8, carried the mutant o2 allele, hence the expression pattern observed was similar to lpa1-1 introgressed progenies having mutant o2 allele.

Expression Pattern of o2 Gene during Kernel Development Stages
The lpa1-1 inbreds with mutant o2 allele and HKI1105 possessing wild-type O2 allele showed differences in transcript levels of o2 gene during different stages of kernel development (Figure 3b). The highest mean expression of o2 gene was observed at 15 DAP (0.067) followed by 30 DAP (0.028), while the lowest expression level was detected at 45 DAP (0.017) across the lpa1-1-based inbreds (Figure 3b). The control inbred HKI1105 showed a similar pattern for o2, with highest expression at 15 DAP and lowest expression at 45 DAP. Among the lpa1-1-based improved lines, the highest expression level of o2 was observed for PMI-PV6-lpa1-A (0.079) at 15 DAP, while the lowest expression level was found for PMI-PV5-lpa1-B (0.012) at 45 DAP. The highest and lowest transcript levels of O2 in HKI1105 were 0.821 and 0.205, respectively. Across the kernel development stages, lpa1-1-based improved lines recorded significantly lower levels of o2 expression (13.07-fold lower transcripts) over the wild-type (O2) in control inbred (HKI1105). The inbreds, viz. PMI-PV5, PMI-PV6, PMI-PV7 and PMI-PV8, carried the mutant o2 allele, hence the expression pattern observed was similar to lpa1-1 introgressed progenies having mutant o2 allele.

Expression Pattern of crtRB1 Gene during Different Stages of Kernel Development
The inbred (HKI1105) with wild-type CRTRB1 allele and lpa1-1-based lines with mutant crtRB1 allele displayed significant differences in the transcript levels during kernel developmental stages (Figure 3c). The mean expression level of crtRB1 among lpa1-1based lines attained highest peak at 15 DAP (0.032), while the lowest expression level was noticed at 45 DAP (0.014). The wild-type CRTRB1 allele in HKI1105 showed a similar trend with highest mean expression level at 15 DAP (0.387) and lowest expression level at 45 DAP (0.209) (Figure 3c). However, the mean expression level was significantly higher in HKI1105 compared to lpa1-1-based inbreds (Figure 3c). The progeny, viz. PMI-PV5-lpa1-B among lpa1-1-based inbreds, showed highest expression of crtRB1 gene at 15 DAP, while PMI-PV6-lpa1-B showed lowest expression at 45 DAP. The lpa1-1-based improved lines recorded significantly lower transcript levels of crtRB1 (12.32-fold) over wild-type CRTRB1 allele present in HKI1105. The lpa1-1-based introgressed lines possessed crtRB1 mutant allele which was also present in PMI-PV5, PMI-PV6, PMI-PV7 and PMI-PV8; hence, the expression levels were comparable among recurrent parents and lpa1-1-introgressed lines, displaying the same expression pattern and mean expression levels (Table 3).  (Table 4; Figure S1a). However, the lpa1-1 expression showed significantly negative correlation with iP accumulation during their respective kernel developmental stages across all wild-type and mutant versions (r = −0.98 to −0.90) ( Figure S1a). Similarly, negative correlation was found for transcript levels of o2 gene with lysine (r = −0.91 to −0.84) and tryptophan (r = −0.92 to −0.85) at various kernel developmental stages ( Figure S1b). Moreover, the expression level of crtRB1 gene possessed negative correlation with proA (r = −0.94) in all three stages of kernel development across the genotypes ( Figure S1c).

Discussion
Phytic acid (PA) is a critical anti-nutritional factor in human and monogastric animals as negatively charged phosphorous in PA chelates positively charged minerals and makes them unavailable in the gut [11,28]. In addition, traditional maize is poor in proA and essential amino acids such as lysine and tryptophan [27]. Here, we investigated accumulation pattern of PA, proA, lysine and tryptophan, and expression of lpa1-1, crtRB1 and o2 genes among a set of newly developed unique maize genotypes during different stages of kernel development [29]. The expression profiling of target genes and the study of their relationships with nutrient accumulation provides new insights into their regulation in the biosynthesis pathway during various kernel developmental stages [30].

Expression Pattern of lpa1-1 and Its Effect on Kernel Phytate Accumulation
Comparing controls with wild-types and lpa1-1-based inbreds, we found that the transcript levels in the wild-types were 4.1-fold higher. These comparisons also showed a strong positive relationship of lpa1-1 transcript levels with accumulation of PA and PA/TP ratio. Low PA and PA/TP ratio signified the higher bioavailability of minerals as compared to wild-type inbreds [24,25]. The lpa1-1 mutant was developed through ethyl methanesulfonate (EMS) at United States Department of Agriculture (USDA) [18]. Mutation is generated due to distortion in a trans-membrane transporter protein (ZmMRP4) gene that encodes for membrane transport protein and helps in transport of PA to storage vacuole [31]. The defective transporter protein is unable to store PA inside protein-storage vacuoles [31]. The maximum expression of lpa1-1 was observed at 15 DAP, while the least expression was observed at 45 DAP. However, the maximum concentration of PA was observed at 45 DAP and minimum at 15 DAP among lpa1-1-based improved lines. In the case of iP, the majority of lpa1-1-based lines showed maximum concentration accumulated at 15 DAP and minimum accumulation at 45 DAP. PA/TP ratio showed lowest percentage at 15 DAP and highest at 45 DAP.

Expression Pattern of o2 and Its Effect on Lysine and Tryptophan
The lpa1-1-based maize inbreds possessed significantly higher lysine (2.27-fold) and tryptophan (2.17-fold) compared to the wild-type (O2) control. In this study, the lpa1-1based inbreds were found to have 13-fold reduced expression of the o2 allele in comparison to the wild-type O2 allele in control inbred. Additionally, negative correlation was observed between o2 transcript levels and accumulation of amino acids (lysine and tryptophan). The leucine zipper family transcriptional factors, which are necessary for the synthesis of 22-kDa-α-zeins (which lack lysine), is encoded by the O2 gene [32]. The o2 mutant gene is responsible for enhancing the synthesis of lysine-rich non-zein protein rich in lysine and tryptophan in addition to negatively regulating lysine keto-reductase [33]. The highest mean expression of o2 gene was observed at 15 DAP, while the lowest mean expression was observed at 45 DAP. The decreasing pattern of amino acids (lysine and tryptophan) with kernel maturity indicates that delay would lead to the loss of nutritional quality [32,34]. As moisture content in maize kernels decreases, genes involved in the production of micronutrients are prevented from being transcribed [35]. This indicated that higher nutritional quality could be achieved when cobs are harvested as green cobs rather than at the dried stage.

Expression Pattern of crtRB1 and Its Effect on proA Accumulation
In the current study, lpa1-1-based inbreds with mutant crtRB1 allele had significantly higher proA (4.3-fold) over those having wild-type CRTRB1 allele. The lpa1-1 inbreds had 12.32-fold lesser transcript levels than the wild-type inbred carrying CRTRB1 allele at various stages of kernel development. The favourable crtRB1 allele enhances the proA content by partially blocking the hydroxylation stages in the branch of the carotenoid biosynthesis pathway [36]. This was additionally supported through significant negative correlation between the levels of crtRB1 transcripts and accumulation of proA carotenoids [37]. The highest expression of crtRB1 was observed at 15 DAP, while the lowest expression level was at 45 DAP. Maize kernels gradually lose moisture content as they mature, which lowers the transcription of several genes associated with vital nutrients. This suggested that green cobs harvested at dough stage would provide higher proA than those harvested as dried stage.
The study clearly suggests that the maize genotypes developed and evaluated in the present study would offer highly bioavailable phosphorous which would help in growth and development of bones and also prevents eutrophication. In addition, the inbreds rich in lysine, tryptophan and provitamin-A would help in developing maize hybrids for their future use in providing higher protein quality and vitamin-A.

Field Experiments
A set of 13 selected inbreds with (i) mutant lpa1-1, o2 and crtRB1 alleles, (ii) wild-type LPA1 allele and mutant o2 and crtRB1 alleles, and (iii) wild-type LPA1, O2 and CRTRB1 alleles, were evaluated at ICAR-IARI, New Delhi (29 • 41 52.13" N, 77 • 0 24.95" E) during the rainy season (July-October) of 2022. Two replications of each entry were raised in one row of 3.0 m length following randomised complete block design (RCBD) with row-to-row and plant-to-plant spacing of 75 cm and 20 cm, respectively. Standard agronomic practices were followed to raise the crop as described in earlier studies [33]. Three to four plants in each of the genotypes per replication were selfed to avoid xenia effects caused due to foreign pollens. The selfed-ears were harvested at 15, 30 and 45 days after pollination (DAP) from each replication. The selfed-ears were stored at −80 • C until RNA isolation and determination of quality parameters.

Isolation of RNA and cDNA Synthesis
RNA was isolated from kernels following a modified version of the traditional RNA Trizol technique as per Dutta et al. [38]. RNA quality was checked using 0.8% agarose gel with 1% formaldehyde and Nabi UV/Vis Nano Spectrophotometer by determining the ratio of absorbance at 260/280 nm. The total RNA was converted to cDNA through reverse transcription using Verso cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Lithuania, Vilnius). For each reaction, 1 µL of Verso Enzyme Mix, 2 µL of dNTP mix, 1 µL of anchored oligo-dT, 1 µL of RT enhancer, 4 µL of 5X cDNA synthesis buffer, 5 µg of template (RNA) and nuclease-free water were combined to make a 20 µL reaction mixture. BIO-RAD T100 TM thermal cycler (Bio-Rad Laboratories, Inc., Singapore) was used for incubation to convert to cDNA at 42 • C for 60 min followed by 95 • C for 2 min. A UV-spectrophotometer was used to measure the concentration of cDNA samples.

Designing of Primers for the Expression Studies
Primer3 Input v4.1.0 software was used to design the primers for expression of lpa1-1, o2, crtRB1 and Adh1 (endogenous control) with GenBank accession numbers EF586878, X15544, GQ889716 and NC 050096.1, respectively. lpa1-1 is a mutant of Lpa1 gene that encodes for a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter responsible for transporting phytic acid to the storage vacuole [31]. In maize kernels, CrtRB1 encodes for β-carotene hydroxylase, which converts αand β-carotene into non-provitamin A carotenoids, viz. lutein and zeaxanthin [36]. Leucine-zipper (bZIP) protein, which is produced by the O2 gene, functions as a transcriptional factor for development of the zein family of storage protein genes, particularly 22 kDa α-zein in maize [39]. Alcohol dehydrogenase 1 (ADH1) is a constitutive gene that aids in cell survival in low oxygen environments (anaerobic conditions) [40]. Details of the expression primers used are given in Table S1. The primers were synthesized by M/S Macrogen Pvt. Ltd., Seoul, Korea.

Samples Preparation for Biochemical Analysis
Part of the self-pollinated ears (harvested at 15, 30 and 45 DAP) remaining after RNA isolation were shelled and left to dry for a further period of 72 h at room temperature in a silica gel-filled container [41]. Randomly selected kernels (n = 100) were taken and kept in desiccator with silica gel particles replaced in every 24 h to keep the kernel moisture content below 15%. The container was completely covered, and the kernels were wrapped in blotting paper using aluminium foil to avoid deterioration brought on by light compounds. The drying process was carried out at a temperature of 25 • C and the dried kernels were processed into a fine powder in the dark using a coffee grinder (Philips, HL7756) machine. Samples from each of the genotypes were reduced to 5 g using quartering technique and stored at −20 • C for further analysis.

Determination of Phytic Acid (PA) and Inorganic Phosphorous (iP)
A modest modification of the protocol published by Lorenz et al. (2007) was followed for the determination of PA and iP [42]. In 2 mL micro-centrifuge tube, 100 mg of maize kernel powder from each of the samples was added along with 2 mL of 0.65 M HCl. The tubes were then placed on a shaker with 200 rpm for an overnight period at room temperature and then centrifuged for 5 min at 10,000 rpm. From the extract of each of the samples, 500 µL was transferred to a new 15 mL tube for iP quantification and another 500 µL to a 2 mL micro-centrifuge tube for quantification of PA. Quantitative standards for PA and iP were used in equal amounts. KH 2 PO 4 (HiMedia, Thane West, Maharashtra, India) and phytic acid dodecasodium salt from maize (Sigma-Aldrich Chemicals Private Ltd., Industrial Area, Bangalore, India) were used as iP standard and PA standard, respectively. Reagent for the iP quantification was prepared right before use and contained in the ratio of 2 (double distilled H 2 O): 1 (ammonium molybdate (0.02 M)): 1 (sulphuric acid (3 M)): 1 (ascorbic acid (0.57 M)). For the determination of iP, each sample received one ml of reagent and one ml of double-distilled water. The optical density was measured at 820 nm after 15 min of incubation at room temperature when the blue colour had fully developed. In order to determine the amount of PA, 1.25 mL of the wade reagent was added to each micro-centrifuge tube. The pink colouration developed after an incubation period of 15 min under room temperature and optical density was measured at 490 nm. The wade reagent consisted of 80 mL of double-distilled water, 0.03 g of FeCl 3 ·6H 2 O and 0.3 g of 5-sulfosalicylic acid. The aforementioned solution was chilled overnight, and the following day, it was treated with NaOH to bring its pH level to 3.05 and volume made up to 100 mL. PA was divided by conversion factor 3.55 to convert to phytate phosphorous (P) [24]. Proportion of PA in the kernel was determined as the ratio of phytate (PA)/TP, where TP = PA + iP, and the ratio was converted to percent.

Determination of Lysine, Tryptophan and Provitamin-A
Dried kernels from stored kernels were analysed for lysine, tryptophan and proA using UHPLC system (Ultra High-Performance Liquid Chromatography; Thermo Scientific, Massachusetts, USA) [43,44] Thermo Scientific's Acclaim-120 C 18 (5 µm, 120Å, 4.6 × 150 mm) column was used to separate samples of lysine and tryptophan, which were then analysed using a diode array detector-3000 (RS) with absorbance at 265 nm and 280 nm, respectively. By comparing the area of the amino acid mix standard with the area of the sample, the concentration of lysine and tryptophan present in the sample were determined. To avoid degradation due to oxidation by light, carotenoids extraction was carried out in the dark, using a modified version of a previously reported procedure [45]. Specimens were taken using YMC Carotenoid C 30 column (5 m, 4.6 × 250 mm) through UHPLC system (Thermo Scientific, Walthamm, MA, USA). A diode array detector-3000 (RS) with absorbance at 450 nm was used to detect β-carotene (BC) and β-cryptoxanthin (BCX). ProA concentration was calculated as the sum of BC and 50% of BCX [27].

Statistical Analysis
Mean nutritional quality traits and transcript levels at different kernel developmental stages were considered for analysis of variance (ANOVA) using 'anova' function in 'R v4.2.1 statistical tool. The correlation plots were deduced using 'metan' package in 'R v4.2.1 . The bar plots were constructed using Microsoft Office-Excel 2019.

Conclusions
Phytic acid in maize acts an anti-nutritional factor reducing the bioavailability of mineral nutrients. The results revealed that the introgression of favourable allele of lpa1-1 led to significant reduction of PA and PA/TP ratio and enhancement in iP. Favourable o2 allele led to enhancement in lysine and tryptophan, while crtRB1 introgression resulted in enhancement in proA. The transcript levels of lpa1-1, o2 and crtRB1 genes were significantly lower than wild-type alleles (LPA1, O2 and CRTRB1). The highest reduction of PA was observed at 15 DAP which also had the highest accumulation of proA, lysine and tryptophan. The transcript levels of lpa1-1 gene showed positive correlation with PA, while negative correlation was found for o2 with lysine and tryptophan, and crtRB1 with proA. This is the first comprehensive study on understanding the regulation pattern of lpa1-1, o2 and crtRB1 genes and their relationship with accumulation of PA, iP, PA/TP, lysine, tryptophan and proA at different stages of kernel development among lpa1-1-based maize genotypes.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12091745/s1, Figure S1: Correlation between gene expression and nutrient accumulation at 15, 30 and 45 days after pollination (DAP); Table S1: Details of primers used for quantitative real-time PCR analysis; Table S2: Concentration of kernel phytic acid (PA) and inorganic phosphorous (iP) among the genotypes; Table S3: Concentration of kernel lysine, tryptophan and provitamin-A contents among the genotypes.