Temporal profile of amino acids and protein fractions in the developing kernel of maize germplasm | Scientific Reports

Scientific Reports volume 14, Article number: 27161 (2024) Cite this article

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Maize, the most important source of animal and poultry feed, is deficient in essential amino acid methionine. Therefore, methionine is added to the poultry feed to meet its nutritional requirements. Keeping in view, an urgent requirement exists to develop high-methionine maize. The present study was designed to understand the synthesis and accumulation pattern of methionine, lysine, tryptophan, total protein, and protein fractions in the developing maize kernel. Results revealed that methionine accumulation starts before 15 DAP and increases towards maturity. Total protein, albumin, and globulin accumulation showed a declining trend, whereas, prolamin, prolamin-like, glutelin, and glutelin-like fractions increased with kernel maturity. Methionine showed a significant positive correlation with prolamin and a negative correlation with glutelin, indicating their use as markers to select high methionine lines. Higher level accumulation of lysine, tryptophan, and methionine, the three essential amino acids deficient in maize, was observed highest in lines 174705 and 194010 indicating their use as a potential donor for developing high methionine maize genotypes. The high methionine line identified in the present study can be used in breeding programs through introgressing maize germplasm of diverse genetic backgrounds to develop high-yielding methionine-rich maize genotypes to develop a sustainable nutritive feed supply chain.

Maize is a globally important crop known as the “Queen of Cereals” due to the highest genetic yield potential among cereals. The crop has tremendous genetic variability, which enables it to thrive in tropical, subtropical, and temperate climates. The worldwide production of maize was around 1123.07 million metric tonnes (M MT) in 2020–2021. Maize is the third most important cereal crop after rice and wheat in India. Total maize production accounts for ~ 9 percent of total food grain production in the country. India contributes around 2.80% of maize production with a quantum of 31.51 M MT in 2020–20211. Maize is a staple food in Africa, South America, and some parts of Asia. In India, about 60% (18.91 M MT) of maize produced is used for livestock and poultry feed, 20% (6.30 M MT) as food, and the rest is used as fuel (3.0 M MT) and for industrial purposes (3.2 M MT)2.

Structurally, the maize kernel consists of an embryo (12%), a much larger endosperm (82%), and a pericarp (6%)3. Maize endosperm contains ~ 70% starch and 10% protein4. Endosperm storage proteins are classified as albumins (3%), globulins (3%), prolamins (also known as zein) (60%), and glutelins (34%). Zeins are further classified based on their molecular weight and genetic properties: α-zeins (19 and 22 kDa), β-zeins (15 kDa), γ-zeins (50, 27, and 16 kDa), and δ-zeins (18 and 10 kDa)5,6. The α-Zein is the most abundant fraction, amounting to 70–85% of the total zein, followed by γ- (10–20%), β- (1–5%), and δ-zein (1–5%)7. All zein fractions are amphiphilic since they contain both hydrophobic and hydrophilic amino acids.

Methionine is an important amino acid required for poultry feed as it plays important roles in many cellular processes. A significant increase in the weight of chicks and egg production has been reported by increasing the quantity of methionine in the diet8,9. However, methionine was limited in all feed resources8. Di Buono et al.10 reported that the mean requirement for methionine is 12.6 mg/kg/day for humans. Plants, unlike animals, synthesize methionine de novo and are thus used as natural dietary sources. Wide variation exists for methionine among different plant species. Maize is a poor source of methionine as normal maize contains < 0.8% methionine therefore, supplementation of poultry feed with synthetic methionine is a common practice. Synthetic amino acids have become a multi-million-dollar industry11. The total worldwide methionine market was 685–700 million tonnes in 2017 which increased by 27% in 2018. The world market of synthetic methionine for livestock and poultry feed is expected to reach US$5.1 billion by 202412. Supplementation of essential amino acids increases the cost of feed, particularly in the case of tryptophan and methionine for which inexpensive supplements are lacking13.

Maize grain protein is nutritionally imbalanced due to a deficiency of essential amino acids, methionine, lysine, tryptophan, and threonine in the prolamin fraction, which is the highest proportion in the storage proteins13. Among zeins, α- and γ-zeins are the most abundant proteins, but are deficient in methionine (sulfur-containing amino acid)3. However, minor zeins, viz., β-, and δ-zein are rich in sulfur-containing amino acids and vary in expression levels among maize genotypes. The δ-zeins are rich in methionine (22%), whereas the γ-zeins are abundant in cysteine (7%); β-zein has high percentages of cysteine (4%) and methionine (11%), while α-zeins lack both of them14. Therefore, maize lines with high δ-zein can be a potential source to replace synthetic methionine in regular feed15, as the majority of the maize produce is consumed as poultry feed. Although, a corn-legume mixture is used to meet the lysine and tryptophan requirements, this mixture remains deficient in methionine. The proportion of methionine, lysine, and tryptophan is more important than the total protein content in any animal feed mixture. There is a prime need to develop maize varieties rich in methionine, lysine, and tryptophan, so as to reduce or replace the use of synthetic amino acids and avoid this cost. Therefore, it is concluded that bio-fortification of maize is an important area of basic and applied research. Various studies are conducted to understand the accumulation pattern and regulation of methionine biosynthesis but very little is known about the regulation of the metabolic pathways for amino acid synthesis in maize16. Additionally, different strategies were applied for the enhancement of methionine percentage in maize12,17. Newell et al.18 reported that targeted enhanced expression of methionine-rich proteins is linked with a reduced level of cysteine-rich γ-zein (27 kDa) thus reducing kernel hardness. 27 kDa γ-zein plays an important role in kernel texture as it maintains kernel hardness and vitreousness. Reduced level of 27 kDa γ-zein results in a soft and chalky kernel which is more prone to mechanical damage, yield loss, and susceptibility to insect and fungal attacks. Whereas, the development of QPM with high lysine, tryptophan, and vitreous kernel texture depends on the increased expression of 27 kDa γ-zein19. So, the overall proteome reframing is needed to increase the essential amino acids viz., methionine, lysine, and tryptophan without compromising kernel texture and agronomic performance.

The present study was designed to understand the synthesis and accumulation pattern of essential amino acids viz., methionine, lysine, tryptophan, protein, and protein fractions during kernel development in maize. The information emanating from it will optimize the nutritional quality of developing maize kernel for its use at appropriate stages for maximum nutritive benefits. The outcome will also provide a correlation among amino acids and protein fractions, which will be helpful in understanding the accumulation pattern of amino acids, total protein, and protein fractions and offer potential solutions for enhancing the overall nutritional value of maize.

To understand the synthesis pattern of methionine and correlation of various parameters viz., methionine, lysine, and tryptophan, total protein and protein fractions (albumin, globulin, prolamin, prolamin-like, glutelin, and glutelin-like), were estimated at 15, 30, and 45 DAP in the developing maize kernel of the experimental genotypes. Protein content ranges from 9.36% (E7-11–1) to 18.86% (VS-5) at 15 DAP, 9.26% (E7-64) to 14.89% (VS-5) at 30 DAP, and 7.58% (VS-64) to 14.89% (E7-24–3) at 45 DAP. Significant differences were observed in the protein content and high genetic variability was observed among different maize genotypes. Protein content showed a declining trend with kernel development as maximum protein was observed at 15 DAP (13.77%), intermediate concentrations at 30 DAP (12.20%), and minimum protein content at 45 DAP (11.80%) (Table 1). Along with this, the hundred kernel weight of these lines is presented in the previous report of Devi et al.20 which showed that kernel weight ranged from 9.42 g to 31.86 g. Minimum kernel weight was observed in E11-35 (9.42 g) and maximum grain weight was recorded in the experimental line E9-25–1 (31.86 g). Correlation of maize inbred lines revealed that hundred kernel weight showed a significant positive correlation (0.91) with methionine content20.

The maize kernel protein is traditionally classified as albumin, globulin, prolamin, prolamin-like, glutelin-like, and glutelin based on its solubility in different solvents as described by Osborne21 and later modified by Landry and bureaux22. Percentage of protein fractions are responsible for different kernel textures and variation in phenotype of maize kernel is depicted in Fig. 1. Normal maize contains higher zein percentage and thus its kernel texture is hard whereas opaque kernels have lower zein in comparison to normal grain. Albumin and globulin content decreased from the initial to the final stage of kernel maturity. Maximum albumin content was present at 15 DAP (14.46%), and minimum at 45 DAP (9.77%). It ranges from 11.69% (174705) to 15.94% (E7-15–2-1–1-1) at the 15 DAP, 8.19% (E2-52–1) to 14.44% (VS-64) at the 30 DAP, and 6.63% (E4-5–2) to 12.75% (VS-26) at 45 DAP indicating the presence of natural variability among maize genotypes. Albumin content was higher in low methionine lines as compared to high methionine lines (Table 1). Albumins showed a partial negative correlation with methionine as it contains only 2.7% of methionine (ExpasyprotParam tool). Globulin content varied from 8.0% (E19-14–1) to 10.19% (E16-25-B-2) at 15 DAP, 7.85% (E19-14–1) to 9.63% (E2-7) at 30 DAP and 7.0% (E7-15–2-1–1-1) to 9.06% (E2-7) at 45 DAP. Maximum globulin was recorded at 15 DAP (9.40%), average at 30 DAP (8.86%), and minimum content was observed at 45 DAP (8.27%). The average content of globulin was higher in low methionine; 9.40%, 8.90%, and 8.59% as compared to high methionine lines; 9.40%, 8.84%, and 8.01% at 15, 30, and 45 DAP, respectively (Table 1). A significant difference exists between experimental lines and between different stages of kernel development for albumin and globulin content.

Screening of maize kernel on a lightbox for its kernel phenotyping.

The major endosperm storage protein, the prolamin and prolamin-like fractions, are collectively referred to as zeins. Prolamin content increased from the initial stage towards kernel maturity, irrespective of the experimental lines. Minimum (average of all the lines) prolamin was observed at 15 DAP (19.93%), intermediate content at 30 DAP (28.69%), and maximum content was found at 45 DAP (36.79%). It varied from 11.75% (VS-64) to 28.13% (174705) at 15 DAP, 13.88% (E4-14–1) to 38.13% (174705) at 30 DAP, and 22.19% (E4-14–1) to 47.44% (194010) at 45 DAP. Average prolamin content was higher in high methionine lines; 21.18%, 33.04%, and 40.84% and low in low methionine lines; 18.42%, 23.46%, and 31.93% at 15, 30 and 45 DAP, respectively. Prolamin-like fraction showed a similar pattern as an increasing trend was observed from the initial stage towards kernel maturity. This fraction varied from 7.0% (E2-52–1) to 11.56% (E9-25) at 15 DAP, 8.81% (E19-22–2) to 12.63% (E2-7) at 30 DAP, and from 10.31% (E17-1–13) to 13.88% (E2-7) at 45 DAP. Minimum average content (9.77%) was observed at 15 DAP, intermediate (10.97%) at 30 DAP, and maximum content (11.90%) at 45 DAP. A higher content of zein fractions maintains the hardness and vitreousness of maize kernel and results in lower insects and fungal attacks. Average prolamin-like content was low at 15(9.48%) and 30 (10.57%) DAP and higher at 45 DAP (12.00%) in high methionine lines, whereas, it remains the same throughout kernel development in low methionine lines (Table 1).

Glutelin is another major protein fraction that showed increasing accumulation with kernel maturity. Average glutelin content was minimum (9.68%) at 15 DAP, intermediate (12.19%) at 30 DAP and maximum (15.08%) at 45 DAP. Glutelin content varied from 2.05% (E2-52–1) to 19.25% (VS-64) at 15 DAP, from 3.42% (E7-11–1) to 20.19% (VS-64) at 30 DAP, and from 9.44% (E17-1–13) to 21.88% (E9-25) at 45 DAP. Comparison of average glutelin fraction showed that low methionine lines had higher glutelin content at 15, 30, and 45 DAP respectively, and thus soft and chalky kernel texture in comparison to high methionine lines.

Similar to glutelin, glutelin-like fraction also showed an increasing trend from 15 to 45 DAP. It varied from 6.44% (E17-1–13) to 24.63% (E2-7) at 15 DAP, from 9.63% (194010) to 34.25% (E19-22–2) at 30 DAP, and from 12.81% (194010) to 38.75% (E4-5–2) at 45 DAP. Similar contents were observed for glutelin-like fractions between high and low methionine lines at 15 and 30 DAP (Table 1). However, a clear difference at 45 DAP was observed as low methionine lines had higher glutelin-like content as compared to high methionine lines.

Although the accumulation of methionine is much less at the initial stage of kernel development (15 DAP), it increased significantly at the intermediate stage (30 DAP) and remained constant until kernel maturity. Maximum methionine was reported in the genotypes 174705 and 194010 having 3.29 and 2.27% of methionine per 100 g protein, respectively. The lowest methionine content was observed in lines E4-14–1 (0.31) and E9-25 (0.30) (Table 2).

Tryptophan and lysine are the other limiting amino acids coded by non-zein proteins. Tryptophan content was higher at 15 DAP, maintained till the intermediate stage, and increased at kernel maturity. It varied from 0.50% to 1.24% at 15 DAP, from 0.35% to 1.34% at 30 DAP, and from 0.61% to 2.23% at 45 DAP among experimental lines. The highest amount of tryptophan was recorded in E7-64 having 2.229%/100 g of protein. The lowest tryptophan was observed in E7-24–3 and E8-16–1-2 both having 0.65%/100 g of protein (Table 2).

A similar trend was observed for lysine accumulation. Lysine concentration was higher at the early stage of kernel development (15 DAP). As the kernel development proceeds, lysine content decreases. Amongst the experimental lines, it varied from 0.97 to 5.95% at 15 DAP, from 2.08 to 11.59% at 30 DAP, and from 1.20 to 5.49% at 45 DAP. The highest lysine content was observed in E7-64 and E4-5–2 having 5.48 and 5.49% of lysine/100 g protein, respectively, whereas the lowest lysine content was observed in E7-24–3 (1.47%) and VS-64 (1.2%) (Table 2).

The data showed the comparison of methionine, tryptophan, and lysine content at different stages of kernel development. Two lines each with the highest and lowest methionine content were selected to compare the synthesis pattern of these amino acids. Methionine content was recorded to be much higher in high methionine lines at all the stages of kernel development in comparison to low methionine lines. Tryptophan and lysine content were slightly lower in high methionine at the final stage of seed maturity in comparison to low methionine lines, indicating that methionine is negatively correlated with both lysine and tryptophan. The fold change at 45 DAP in the experimental line 194010 has 2.12, 0.39, and 0.37 in methionine, tryptophan, and lysine, respectively. Methionine in line 174705 showed a fold increase up to 0.82 on the 30 DAP and 2.83 on the 45 DAP. Methionine showed a fold increase of 1.56, whereas, tryptophan and lysine showed a fold increase of 0.34 and 0.40 at 30 DAP, respectively, indicating a combined accumulation of lysine and tryptophan along with methionine in this particular genotype. For the comparative study of amino acids, the average of these amino acids in high and low methionine lines is calculated separately and utilized for the evaluation of the best maize genotype and developmental stage (Fig. 2).

Average amino acid percentage in high and low methionine lines during kernel development. Error bar represents standard error. Tukey’s test (P < 0.05): Letters A, B, C… and so on depict that sample means are significantly different from each other.

The dendrogram analysis of the experimental genotypes conducted based on group average, squared Euclidean method for the above parameter is depicted in Figs. 3, 4 and 5. Numbers 1–12 represent high methionine lines whereas, 13–22 represent low methionine lines. Overall, the dendrogram analysis showed that the prolamin and glutelin fractions differentiate the high and low methionine lines. So, these fractions can be used as potential protein markers to identify the high methionine maize lines. The analysis for protein revealed that methionine percentage does not affect the protein content at any stage of kernel development, as no considerable differences were observed between experimental lines (Fig. 3). No differentiation was observed among the experimental lines for methionine (Fig. 4a), tryptophan (Fig. 4b), and lysine at any stage of kernel development (Fig. 4c). Albumin, globulin, prolamin-like, and glutelin-like fractions did not show any differentiation among experimental lines (Fig. 5a,b,d,f), whereas the prolamin content clearly differentiates high and low methionine lines at kernel maturity (Fig. 5c). It also proved that the prolamin fraction is associated with methionine and is a major key factor in increasing methionine content in maize. Similarly, glutelin fraction differentiates high and low methionine lines at all the stages of kernel development (Fig. 5e).

Dendrogram of protein content at different stages of kernel development.

Dendrogram of methionine (a), tryptophan (b) and lysine (c) content at different stages of kernel development.

Dendrogram of albumin (a), globulin (b), prolamin (c), prolamin-like (d), glutelin (e) and glutelin-like (f) content at different stages of kernel development.

Correlation of proteins, protein fractions, and amino acids Correlation analysis showed that at 15 DAP albumin and prolamin content showed a significant negative correlation with each other (r = − 0.41), and methionine showed a significant negative correlation with glutelins (r = − 0.52). At 30 DAP, methionine showed a significant positive correlation with prolamin content (r = 0.72) but a significant negative correlation with glutelin content (r = − 0.89). Prolamin showed a significant negative correlation with glutelin (r = − 0.70). At 45 DAP, glutelin showed a significant negative correlation with protein (r = − 0.50), methionine (r = − 0.78), and prolamin (r = − 0.77) but a significant positive correlation with globulin (r = 0.50). The protein showed a significant positive correlation with prolamin (r = 0.44), and albumin showed a significant negative correlation with prolamin-like (r = − 0.38) (Fig. 6).

Correlation of protein, lysine, methionine, tryptophan, and protein fractions at 15 (a), 30 (b), and 45 (c) DAP of kernel development.

The results revealed, a decreasing trend in protein in low methionine lines whereas, no consistent trend was observed in the case of high methionine lines, indicating that methionine-associated protein decreases towards kernel maturity in low methionine lines. Therefore, it is pertinent that methionine-associated proteins are more important than total protein content in developing high methionine lines. High protein content at the early stages of kernel development indicates that actively dividing cells need a continuous supply of proteins, whereas, the kernel becomes metabolically dormant at maturity, and hence protein synthesis is reduced. In mature kernels, tissues are stabilized by accumulating stable storage products, especially starch which lowers the requirement of protein synthesis as compared to the immature stage23. Similar trends in protein accumulation in developing maize endosperm were reported by Wall and Bietz24. Whereas, Ortega et al.25 reported that protein content decreases with kernel development because of increased starch synthesis and enzymatic nitrogen breakdown. A protein accumulation pattern was also reported by Jin et al.26 who described that by 17th to 30 DAP, the number of protein isoforms decreases from 439 to 383 in normal maize. Similar types of results were also reported by Sethi et al.27, showing that protein content decreased from an early stage towards kernel maturity in maize. The higher protein percentage at the early stages of kernel development is the major reason for the nutritional superiority of baby corn, sweet corn, and green maize used for human consumption. It is concluded that the consumption of maize at these stages is a cost-effective way to improve protein nutrition.

It has been reported that zein proteins viz., δ-zein have 22.48% and β-zein have 11.25% of methionine, whereas non-zeins are poor in methionine content14. Zein fractions (Prolamin and prolamin-like) showed an increasing trend from the initial stage toward kernel maturity. Prolamin-like fraction accumulate at a higher rate in the methionine-rich line, indicating its direct correlation with methionine, a novel key finding of the present study. Prolamin-like content was mainly distinguished at the maturity stage (45 DAP). Prolamin and prolamin-like together will be used as markers to distinguish high methionine lines. Albumin and globulin proteins decreased towards kernel maturity as they are required at an early stage in actively dividing cells27. The concentration of zein was lower at the initial stage of kernel development and increased with kernel maturity27. Simultaneously, the methionine content also increased towards kernel development. Similar results were reported by Yang et al.28 that prolamin fraction increased with kernel maturity.

Zeins are the most abundant proteins of maize endosperm which maintains kernel hardness and vitreousness, so their expression levels are also high in kernel endosperm27,29. Zein-specific sequences accounted for nearly 50% of the total cDNAs in developing maize kernel29. Transcriptome analysis of the developing maize kernel showed that zein genes accounted for about 65% of the transcripts during the kernel developmental stage of 10–34 DAP30. Glutelin contains only 1.4% methionine, so high glutelin content is an indication of lower methionine concentration in maize. Glutelin and glutelin-like fractions showed significant differences among experimental lines and different kernel developmental stages. Similar results were also reported by Sethi et al.27, indicating that glutelin and glutelin-like content increased from the initial to the final stage of kernel development. Wolf et al.31 observed that both fractions showed an increasing trend concerning endosperm development. In conclusion, albumin, globulin, glutelin, and glutelin-like content were lower in high methionine lines but prolamin and prolamin-like contents were higher in high methionine lines.

From the above, it is concluded that albumin and globulin proteins start accumulating during the early stage of kernel development and decrease towards kernel maturity. Synthesis of prolamin, prolamin-like, glutelin, and glutelin-like proteins starts in the initial stage and keeps on accumulating towards kernel maturity. Metabolically active proteins such as enzymes and signal molecules present in albumin and globulin fractions, play an important role during the initial actively dividing early phase, so these proteins accumulate during the early stage and decrease towards maturity, a stage of lower metabolic activity27. Major storage proteins of maize seeds are zeins including prolamin and prolamin-like32, so their synthesis increased during kernel development27. Guo et al.33 reported that zein proteins significantly contribute to kernel hardness. Zein proteins are also associated with protein body formation and their accumulation starts from 10 DAP during kernel development34. Like zeins, glutelin, and glutelin-like proteins also increased towards kernel maturity and serve as storage proteins and reservoirs of energy for mature maize kernel27.

Zeins are rich in glutamine, proline, leucine, and alanine but are deficient in the essential amino acids particularly, lysine and tryptophan, and therefore are of relatively poor nutritional quality3,27. However, methionine differs from lysine and tryptophan in several ways. In contrast to tryptophan and lysine, β- and δ-zeins are abundant in methionine-rich lines14. Methionine is one of the limiting amino acids which is mainly coded by the zeins protein (prolamin)14. The zein content was low at the initial stage of kernel development concentration which resulted in lower methionine content. As kernel developmental proceeds, the synthesis of zein proteins increases27 thus the quantity of methionine also increases. A significant difference was observed in methionine content among experimental lines and between different kernel developmental stages.

The amount of tryptophan content decreased from 15 to 45 DAP during kernel development. This may be attributed to the higher accumulation of zein proteins in the later stage of kernel development compared to non-zein proteins. So the synthetic tryptophan is added in the poultry feed which enhances the cost of the feed. Lysine content showed significant differences between lines and different kernel developmental stages. Similar findings have been observed by Wall and Bietz24. A study reported that the activity of lysine-ketoglutarate reductase (LKR) is positively correlated with the accumulation of zein proteins. Zein proteins are lysine deficient so LKR degrades free lysine leading to the formation of amino acids important for accumulation with kernel maturity. Therefore, both free and protein-bound form of lysine is reported to decrease with kernel maturity due to increased activity of lysine degrading enzyme and lysine deficient zein accumulation, respectively29,32,35. It is reported that the lysine and tryptophan content is present in 3:1 in maize, so the value of one amino acid is used for calculating the content of the second, but according to the study, it is not valid at each stage of kernel development. As already mentioned lysine and tryptophan are reported to be negatively correlated with methionine3,27, but in the present study, an experimental line 194010 showed a combined accumulation of methionine, tryptophan, and lysine. The combined accumulation of lysine and tryptophan along with methionine is the novel finding of the present study. In conclusion, the experimental line 194010, could be utilized in future breeding programs for developing nutritionally improved maize hybrids. Fold increase in methionine at different stages of kernel development showed that the 30 DAP is the best stage as it retains the highest methionine, lysine, and tryptophan across genotypes except 174705 and 194010. As maximum genotypes retained the highest methionine content at 30 DAP but due to certain exceptions this aspect is open for further research on a larger subset of genotypes where the complete mechanism of methionine accumulation can be analyzed throughout the development. Maize kernel at this stage is harvest-friendly, and has a better nutritional profile, so consumption at this stage is highly beneficial for nutritional purposes. The higher concentration of essential amino acids observed in the early stages of kernel development proves the nutritional superiority of baby corn, sweet corn, and green maize used for silage making27.

As methionine-rich maize is desirable for poultry feed, so mechanism is needed where high methionine can be differentiated from the low methionine maize, so that the poultry sector can develop value chains for procurement and stocking of the high methionine maize. The dendrogram results revealed that methionine is associated with prolamin and glutelin content, thus, these proteins can be used as a protein marker to select the high methionine lines. As methionine is coded by the minor δ-zeins, the zein fraction (prolamin) is associated (directly correlated), whereas, non-zeins are inversely correlated with the methionine content. Comparison of low and high methionine lines also showed that the high methionine lines retained more zeins fractions whereas the low methionine lines retained higher non-zeins.

From the correlation analysis, it was inferred that as total protein increased during kernel development, prolamin content also increased which resulted in increased methionine concentration. Glutelin content decreased with kernel development and showed a negative correlation with zeins and methionine, which showed that glutelins and methionine were inversely correlated to each other. Glutelin content showed a positive correlation with lysine and tryptophan. These results revalidate that the non-zeins are rich in lysine and tryptophan. In conclusion, increased prolamin content resulted in high methionine but lower concentration of lysine and tryptophan, and increased concentration of glutelin resulted in high lysine and tryptophan but low methionine content.

In conclusion, it is inferred that as methionine is an essential amino acid along with lysine and tryptophan limiting in maize grains. In the past decade, research has been oriented toward the enhancement of lysine and tryptophan through the QPM breeding program. However, methionine content remained obscure in the breeding programs. The present study gave a broad perspective to enhance the overall nutritive quality of maize by focusing on three limiting amino acids viz., methionine, lysine, and tryptophan along with their correlation to different protein fractions. Prolamin and prolamin-like fractions have a positive correlation whereas glutelin and glutelin-like fractions have a negative correlation with methionine. Results revealed that the average fold change of methionine, lysine, and tryptophan was positive thus there is a scope for cumulative improvement of these amino acids. So, the present study suggests a balanced increase in lysine and tryptophan under QPM background, along with enhancement of prolamin required for enhancement of methionine accumulation and for maintaining kernel texture. Dendrogram and correlation analysis showed that prolamin can be used as a positive biochemical marker whereas, glutelin can be used as a negative marker for methionine accumulation. The present study also describes that the 30 DAP is the most promising stage in terms of nutritive value in maize in order to harvest the best nutritive value. The present study also identified a breeding line (194010) rich in methionine, along with lysine and tryptophan which may be used as a potential parent line in developing nutritionally improved maize hybrids. It proves that genetic background also plays an important role in regulating the nutritive value of maize. The high methionine donor identified in the present study can be utilized to diversify the breeding program through introgressing maize germplasm with different genetic backgrounds in order to develop high-yielding methionine-rich maize genotypes in order to develop a stable nutritive feed supply chain.

The diverse genetic material of 300 inbred lines procured from ICAR-Indian Institute of Maize Research, PAU Campus, Ludhiana, and International Maize and Wheat Improvement Center, Mexico (CIMMYT) was initially screened for their methionine content. Based on the data obtaineda set of twenty-two experimental lines, 10 low methionine (≤ 0.8%) and 12 high methionine (≥ 1%) were identified. These experimental lines were sown in a plot size of 100 square meters, in a randomized block design in three replications (2020–2021). A total of 50 seeds of each experimental line were sown in three rows (3-m length). Each plant was self-pollinated. Seed samples were collected at 15, 30, and 45 days after pollination (DAP) for the estimation of methionine, lysine, and tryptophan, protein, and protein fractions. The samples were stored at − 80 °C, for further analysis. Experiments on plants/plant parts must confirm that the use of plants in the present study complies with international, national, and/or institutional guidelines. The pedigree/source of each genotype is listed in Table 3. For kernel phenotyping maize seeds were kept on the lightbox and an image was captured to differentiate the seed texture.

The kernel pericarp was removed, and samples were ground to a fine powder and defatted using petroleum ether. Methionine content was estimated by the method of Gupta and Das36. Firstly, the powdered samples were hydrolyzed using a hydrochloric acid solution. Further, hydrolysate was neutralized with sodium hydro-oxide, then sodium nitroprusside and concentrated Ortho-phosphoric acid were used to measure methionine content. Complete enzymatic (Papain) hydrolysis of protein in defatted samples releases free amino acids from which tryptophan and lysine were estimated in both biological and technical triplicates. Tryptophan content was estimated by the method of Hernandez and Bates37 and lysine content was estimated by the method of Tsai et al.38. Protein fractionations were evaluated by the method of Landry and Moureaux22. Albumin was extracted in water, globulin in NaCl, prolamin in isopropanol 70%, prolamin-like in 70% isopropanol + 0.6% 2-Mercaptoethanol (ME), glutelin-like in borate buffer (0.05 M, pH 10) + 0.6% 2-ME, and Glutelin in borate buffer (0.05 M, pH 10) + 0.6% 2-ME + 0.5% SDS solvents. Total protein and protein fractions were estimated in both biological and technical triplicates using the micro Kjeldahl method39 through an automated nitrogen analyzer after digestion with sulphuric acid and catalytic mixture (potassium sulfate, mercuric oxide, and copper sulfate). The amino acid percentage information was retrieved from the ExpasyprotParam tool. The fold change of amino acids was calculated using the formulas given below:

All the experiments were performed in three technical replicates and data was represented as mean value ± standard deviation. Statistical analysis was done using ANOVA (Analysis of Variance) and Tukey’s post hoc test (P < 0.05) among different experimental lines at various stages of kernel development to determine the significant differences among samples for each parameter (among three technical replicates) through SPSS 21.0 software. Standard errors were calculated and presented in the image and tables. Statgraphics 18 (Statistical Graphics Corp. Manugistics Inc., Cambridge, MA) was used to analyze frequency histograms and descriptive statistics. Dendrogram analysis was performed to describe the genetic distance among experimental lines for each variable. Correlation analysis was done through Pearson Product Moment correlations test using statgraphics 18 software at different stages of kernel development. Amino acid percentages in different types of proteins are evaluated using the ExpasyprotParam tool.

All data generated or analyzed during this study are included in this published article.

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Division of Biochemistry, ICAR-Indian Institute of Maize Research, PAU Campus, Ludhiana, 141004, India

Veena Devi, Mehak Sethi & Dharam Paul Chaudhary

Department of Biochemistry, Punjab Agricultural University, Ludhiana, 141004, India

Charanjeet Kaur

Divison of Plant Breeding, ICAR-Indian Institute of Maize Research, PAU Campus, Ludhiana, 141004, India

Vishal Singh & Ramesh Kumar

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Conception or design of the work: D.P.C., V.D. the acquisition, analysis, or interpretation of data: V.D. have drafted the work or substantively revised it: V.D., M.S., C.K. Material provided: V.S., R.K. Approved the submitted version: D.P.C., V.D., M.S., C.K., V.S., R.K.

Correspondence to Dharam Paul Chaudhary.

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Devi, V., Sethi, M., Kaur, C. et al. Temporal profile of amino acids and protein fractions in the developing kernel of maize germplasm. Sci Rep 14, 27161 (2024). https://doi.org/10.1038/s41598-024-65514-2

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Received: 27 September 2023

Accepted: 20 June 2024

Published: 07 November 2024

DOI: https://doi.org/10.1038/s41598-024-65514-2

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