Response of leaf area ratio, specific leaf weight, grain moisture content and quality traits of spring maize (Zea mays L.) to nitrogen dose and application timing

Introduction

Maize is an important cereal and multifunctional crop in the Poaceae family, widely used for human food, animal and poultry feed, and industrial products such as maize starch, dextrose, maize syrup and maize flakes (Gul et al., 2021) [8] . However, maize productivity and grain quality are often low in nutrient-limited soils, especially under nitrogen deficiency. Nitrogen is one of the most essential plant nutrients and component of a wide range of biological chemicals that play critical roles in photosynthetic activity and agricultural productivity (Gheith et al., 2022) [6] . Nitrogen application significantly improved dry matter, yield, harvest index of maize (Yadete et al., 2024) [29] . Increasing nitrogen levels increased leaf growth parameters such as specific leaf area (SLA), leaf area index (LAI) and leaf area ratio (LAR), which ultimately contribute to higher grain yield (Amanullah et al., 2007 and Oscar et al., 2006) [2,20] . The application of nitrogen at 180 kg ha -1 achieved highest grain moisture content than lower dose (Illes et al., 2025) [13] . Grain protein content is particularly responsive to nitrogen fertilization. Madagoudra et al. (2022) [18] reported that highest protein content was obtained with 120 kg N ha -1 , which was statistically at par with 90 kg N ha -1 . Similarly, Aulakh et al. (2012) [3] observed higher protein content at 175 kg N ha -1 , which remained statistically comparable with 150 and 125 kg N ha -1 . They also recorded maximum oil content and minimum carbohydrate content at 175 kg N ha -1 . Ullah et al. (2015) [27] found that application of 280 kg N ha -1 resulted in significantly higher crude protein and crude fibre compared to lower nitrogen levels. Likewise, Kaur and Kumar, (2017) [15] reported the maximum protein content at 180 kg N ha -1 , which was statistically at par with 150 kg N ha -1 along with higher oil content and lower carbohydrate content. Sharma et al. (2017) [22] also confirmed that increasing nitrogen rates significantly enhanced protein content in maize. Nitrogen application timing significantly influenced on maize productivity and nitrogen use efficiency (Gheith et al., 2022) [6] . The highest specific leaf area (SLA), leaf area index (LAI) and leaf area ratio (LAR) were observed in plots where nitrogen was applied in five split doses, with a larger proportion supplied at later growth stages. While, the lowest SLA, LAI and LAR were recorded in plots that received nitrogen in only three splits, with a greater proportion applied at the time of sowing (Amanullah et al., 2007) [2] . Niaz et al. (2014) [19] reported that maximum leaf area index was obtained with 50% N broadcast-incorporated at seed bed preparation + 50% N top dressed at V 9 + C 2 which was at par with 25% N broadcast-incorporated at seed bed preparation + 75% N top dressed at V 9 + C 2 . Hammad et al. (2013) [10] observed that the highest leaf area index (LAI) and leaf area duration (LAD) were recorded when nitrogen was applied in three equal splits, 1/3 at V 2 stage, 1/3 at V 16 and 1/3 N at R 1 stage. Moreover, the crop attained the maximum crop growth rate (CGR) when nitrogen was applied in three equal portions, 1/3 N at seed bed preparation, 1/3 N at V 6 and 1/3 N at VT stage. Therefore, understanding the response of physiological traits such as leaf area ratio and specific leaf weight along with grain moisture and quality traits to different nitrogen doses and application timings is essential for optimizing nitrogen management in spring maize. The present study was conducted to evaluate the response of leaf area ratio, specific leaf weight, grain moisture content and quality traits of spring maize (Zea mays L.) to nitrogen dose and application timing with the aim of identifying efficient nitrogen management practices for improved productivity and grain quality.

Materials and Methods

A field experiment was conducted during the spring seasons of 2013 and 2014 at the Regional Research Station, Uchani, Karnal, of CCS Haryana Agricultural University. The experimental site is situated at 29° 43′N latitude and 76° 58′E longitude, at an altitude of 245 m above mean sea level. The soil was sandy loam in texture with an initial pH of 8.10 and electrical conductivity (EC) of 0.36 dsm -1 . The experiment was laid out in a strip plot design with three replications. The experiment comprised four nitrogen doses, N 1 (150 kg N ha -1 ), N 2 (165 kg N ha -1 ), N 3 (180 kg N ha -1 ) and N 4 (195 kg N ha -1 ) assigned to the main plots. Four nitrogen application timing were allocated to subplots, S 1 (50% at sowing + 25% at 8-leaf stage + 25% at tassel initiation), S 2 (25% at sowing + 25% at 4leaf + 25% at 8-leaf + 25% at silking), S 3 (20% at sowing + 30% at 6-leaf + 40% at flowering + 10% at grain formation) and S 4 (20% at 2-leaf + 30% at 6-leaf + 40% at tassel initiation + 10% at grain formation). The spring maize variety HQPM-1 was used in the study. Sowing was carried out on 5 March 2013 and 10 March 2014 using a seed rate of 20 kg ha -1 . Seeds were dibbled on dry ridges spaced 60 cm apart with 20 cm plant to plant spacing. Irrigation was applied up to half of the ridge immediately after sowing to ensure adequate soil moisture for proper germination. The gross plot size was 15 m 2 , while the net plot size for recording observations was 11.52 m 2 (3.20 m × 3.60 m). Nitrogen in the form of urea (46% N) was used for the study. Nitrogen was applied at different dose and application timing based on treatments. Recommended dose of nutrients such as phosphorous and potassium at 60:60 kg ha -1 were applied uniformly to all plots at the time of sowing. SSP and MOP were used as a source of phosphorous and potassium in the form of P 2 O 5 and K 2 O. The Leaf area ratio (LAR) and Specific leaf weight (SLW) were calculated at 25-day intervals using standard formula. The details are given below.

Leaf area ratio (LAR)

It is a ratio of leaf area per plant to weight per plant and is expressed in cm 2 g -1 . This was calculated by using the following formula.

Specific leaf weight (SLW)

It is a ratio of leaf area per plant to leaf weight per plant and is expressed in cm 2 g -1 . This was calculated by using the following formula.

Grain moisture content (%) was calculated at harvest stage by using grain moisture meter and it is expressed in percent. Protein content of grains from each plot was determined by estimating the total nitrogen content of the sample by using the method of modified Kjeldahl's method (Jackson, 1973) [14] . The estimated nitrogen was multiplied with conversion factor 6.25 to convert total nitrogen in terms of protein. A representative sample of seeds was taken from each net plot produced for analysis of lysine content (%) and it was determined by using the colorimetric method designed by Tasi et al. (1972, Purdue University) [26] and tryptophan content (%) of grain was estimated by gloxylic acid. The data recorded for different parameters were analysed with the help of analysis of variance (ANOVA) technique (Gomez and Gomez, 1983) [7] for strip plot design using SAS 9.1 software (SAS Institute, Cary, NC). The Tukey procedure was used where ANNOVA was significant and results are presented at 5% level of significance (P = 0.05).

Results and Discussion

Leaf area ratio -LAR (cm 2 g -1 ) Leaf area ratio is the ratio of leaf area to total weight of plant. Results revealed that the doses of nitrogen significantly affected LAR at all the growth stages except at 25 DAS (Table 1). Highest LAR was recorded in plots to which nitrogen was applied in low dose of N 1 (150 kg N ha -1 ) followed by N 2 (165 kg N ha -1 ) at 50, 75 DAS and 100 DAS during 2013 and 2014. Lowest LAR was found in plots to which nitrogen was applied in high dose of N 4 (195 kg N ha -1 ) which was statistically at par with N 3 (180 kg N ha -1 ) at all the crop growth stages during both the years of study. Similar results were reported by DaMatta et al. (2002) [4] who reported that LAR decreased with increase of nitrogen application above optimum level. Similar findings were also reported by Singh et al. (2022) [25] who observed that nitrogen levels significantly influenced growth indices in wheat. Kumar et al. (2024) [16] also reported that nitrogen application significantly increased leaf area index and dry matter accumulation, however the response declined beyond the optimum nitrogen level. Among the nitrogen application timing, the leaf area ratio (LAR) differed significantly at all crop growth stages. The highest LAR was recorded in plots where nitrogen was applied in four unequal splits as S3 (20% at sowing + 30% at 6-leaf + 40% at flowering + 10% at grain formation). This treatment was statistically at par with S 4 (20% at 2-leaf + 30% at 6-leaf + 40% at tassel initiation + 10% at grain formation), but both were significantly superior to the remaining nitrogen application timings during both the years of study. The lowest LAR was observed under S 2 , where nitrogen was applied in four equal splits as 25% at sowing + 25% at 4-leaf + 25% at 8-leaf + 25% at silking. The LAR increased under split nitrogen application may be attributed to better coordination between crop nitrogen demand and nutrient supply, which enhances leaf development, canopy expansion and nitrogen use efficiency. Similar findings were reported in maize where split nitrogen application improved leaf area traits, growth parameters and nitrogen use efficiency compared with single time application (Shrivastava et al., 2024 and Singh et al., 2025) [23,24] . Furthermore, applying nitrogen in delayed or multiple splits promotes better leaf area development and canopy expansion which ultimately enhances leaf area ratio. Specific leaf weight -SLW (cm 2 g -1 leaf weight) Specific leaf weight (SLW) is defined as the ratio of leaf area to leaf weight which indicates the leaf thickness (Table 2). The greater the SLW the lesser will be the photosynthesis in leaf. Analysis of two years of maize data revealed that nitrogen dose significantly influenced SLW at all growth stages except at 25 DAS during both years of investigation. The highest SLW values (197.77 and 200.26 cm² g⁻¹ leaf weight) were recorded in plots receiving the lowest nitrogen dose N1 (150 kg N ha -1 ) followed by N 2 (165 kg N ha -1 ), N 3 and N 4 (180 and 195 kg N ha -1 ). While lowest SLW was observed under the highest nitrogen dose N 4 (195 kg N ha -1 ), which was statistically similar with N 3 (180 kg N ha -1 ) at 50, 75 DAS and 100 DAS during both the years. These results are in agreement with the findings of Fan et al. (2022) [5] who reported that increasing nitrogen availability tends to reduce SLA due to enhanced leaf area expansion relative to biomass accumulation, reflecting the plasticity of leaf traits under varying nutrient conditions. Specific leaf weight (SLW) was significantly influenced by nitrogen application timings at all crop growth stages. The highest SLW was recorded when nitrogen was applied in four unequal splits under S 3 (20% at sowing + 30% at 6-leaf + 40% at flowering + 10% at grain formation), which remained statistically similar to S 4 (20% at 2-leaf + 30% at 6-leaf + 40% at tassel initiation + 10% at grain formation) at all growth stages except at 100 DAS in 2014. While, the lowest SLW was observed under four equal splits in S 2 (25% at sowing + 25% at 4-leaf + 25% at 8-leaf + 25% at silking). Similar findings were reported by Amanullah (2009) [1] who reported that application of nitrogen in four to five splits enhanced leaf area and plant height leading to improved maize productivity. 6.18 S1 (50% at sowing + 25% at 8-leaf stage + 25% at tassel initiation), S2 (25% at sowing + 25% at 4-leaf + 25% at 8-leaf + 25% at silking), S3 (20% at sowing + 30% at 6-leaf + 40% at flowering + 10% at grain formation) and S4 (20% at 2-leaf + 30% at 6-leaf + 40% at tassel initiation + 10% at grain formation)

Grain moisture content (%) Grain moisture content of maize was significantly influenced by nitrogen dose and application timing during both the years (Table 3). Grain moisture content was increased with increasing the nitrogen doses. The maximum moisture content was recorded with N4-195 kg N ha -1 (21.34 and 21.71%) and it was at par with N 3 -180 kg N ha -1 (20.69 and 21.17%) as compared to other dose of nitrogen during 2013 and 2014, respectively. Whereas, minimum grain moisture content (18.73 and 19.01%) was recorded in plots to which nitrogen was applied in lower dose of N 1 (150 kg N ha -1 ) during both the year of study. These findings are consistent with Zhang et al. (2021) [30] who observed that maize grain moisture content varied by 1.9-4.0 % among different nitrogen application rates (0-450 kg N ha -1 ). Similar results were also reported by Guo et al. (2020) [9] . In nitrogen application timing, grain moisture content of maize varied significantly among the treatments. The highest grain moisture content was recorded under S 3 (20% at sowing + 30% at 6-leaf + 40% at flowering + 10% at grain formation), which registered 21.22% and 21.52% during 2013 and 2014, respectively. This treatment was statistically at par with S 4 (20% at 2-leaf stage + 30% at 6-leaf stage + 40% at tassel initiation + 10% at grain formation) which recorded 20.43% and 20.80% grain moisture content during the respective years. The higher grain moisture content under these treatments may be attributed to the availability of nitrogen during later growth stages, particularly during grain formation which supports kernel development and prolongs physiological activity. While the lowest grain moisture content was observed under S1 (50% at sowing + 25% at 8-leaf stage + 25% at tassel initiation), with values of 19.25% and 19.77% during 2013 and 2014, respectively.

Quality traits Lysine, tryptophan and protein content

The quality parameters such as lysine, tryptophan and protein content were significantly influenced by different nitrogen doses (Table 3). The maximum lysine, tryptophan and protein content were recorded with N4 (195 kg N ha -1 ), which was statistically similar to N 3 (180 kg N ha -1 ) but significantly higher than N 1 (150 kg N ha ha -1 ) and N 2 (165 kg N ha -1 ) during both years of investigation. Adequate nitrogen availability enhances amino acid synthesis and protein accumulation in maize kernels, thereby improving grain quality. Similar findings were reported by Madagoudra et al., 2022 [18] ; Sharma et al., 2017 [22] who reported that protein content increased with increasing nitrogen dose. Quality characteristics in maize were reported to be increased with nitrogen supply (Hammad et al., 2011c; Amanullah et al., 2009) [11,1] .

In case of nitrogen application timing, maximum lysine, tryptophan and protein contents were observed when nitrogen was applied in four splits as S 4 (20% at 2-leaf stage + 30% at 6leaf stage + 40% at tassel initiation + 10% at grain formation), which was statistically similar with S 3 (20% at sowing + 30% at 6-leaf + 40% at flowering + 10% at grain formation) but significantly higher than S 1 (50% at sowing + 25% at 8-leaf stage + 25% at tassel initiation) and S 2 (25% at sowing + 25% at 4-leaf + 25% at 8-leaf + 25% at silking). These results corroborated with that of Lindiwe and Erick, (2024) [17] , Madagoudra et al. (2022) [18] , Hitesh and Renu, (2009) [12] reported that application of nitrogen in split doses increased grain protein content. Rizwan et al. (2003) [21] and Weiser and Seilmeier (1998) [28] found that the nitrogen fertilizer application timing influenced grain protein content.

Conclusion

The present study indicated that nitrogen dose and application timing significantly affected leaf area ratio (LAR), specific leaf weight (SLW), grain moisture, lysine, tryptophan and protein content. Among nitrogen doses, N 1 (150 kg N ha -1 ) recorded higher LAR and SLW, while the lowest values were observed under N 4 (195 kg N ha -1 ). However, grain moisture content and quality traits such as lysine, tryptophan and protein content were higher with N 4 (195 kg N ha -1 ) and N 3 (180 kg N ha -1 ). In case of nitrogen application timing, S 3 (20% at sowing + 30% at 6-leaf + 40% at flowering + 10% at grain formation) and S 4 (20% at 2-leaf + 30% at 6-leaf + 40% at tassel initiation + 10% at grain formation) treatments produced higher LAR, SLW and improved grain quality traits. Lower values of these parameters were recorded under S 2 (25% at sowing + 25% at 4-leaf + 25% at 8-leaf + 25% at silking) and S 1 (50% at sowing + 25% at 8leaf stage + 25% at tassel initiation). Based on the results, application of 180 kg N ha -1 along with split application as S 3 (20% at sowing + 30% at 6-leaf + 40% at flowering + 10% at grain formation) or S 4 (20% at 2-leaf + 30% at 6-leaf + 40% at tassel initiation + 10% at grain formation) is recommended for improving grain quality traits.

Acknowledgement

AcknowledgmentThis work is part of my Ph. D research (K. C. Verma), I gratefully acknowledge all the authors whose valuable work has contributed to the preparation of this manuscript and supported this study. I am sincerely thankful to CCS Haryana Agricultural University for providing the necessary infrastructural facilities and support during the course of my research.

References

1. Amanullah, Khan KB, Shah P, Maula N, Arifullah S. Nitrogen levels and its time of application influence leaf area, height and biomass of maize planted at low and high density. Pak J Bot. 2009;41:761-768.
2. Amanullah, Hassan MJ, Nawab K, Ali A. Response of specific leaf area (SLA), leaf area index (LAI) and leaf area ratio (LAR) of maize (Zea mays L.) to plant density, rate and timing of nitrogen application. World Appl Sci J. 2007;2(3):235-243.
3. Madagoudra YB, Ardeshna RB, Terin P, Sai Srujanand C, Chaithanya J. The effect of rate and time of nitrogen application on quality of summer maize (Zea mays L.). Adv Biores. 2022;13(2):136-139.
4. Aulakh GS, Vashist KK, Sharma S, Mahal SS. Productivity, quality and water expense efficiency of late kharif sown hybrid maize (Zea mays L.). J Crop Weed. 2012;8(2):139142.
5. DaMatta FM, Loos RA, Silva EA, Loureiro ME. Limitations to photosynthesis in Coffea canephora as a result of nitrogen and water availability. J Plant Physiol. 2002;159:975-981.
6. Fan P, Ming B, Anten NPR, Evers JB, Li Y, Li S, et al. Plastic responses of leaf traits to N deficiency in fieldgrown maize. AoB Plants. 2022;14(6):1-10.
7. Gheith EMS, El-Badry OZ, Lamlom SF, Ali HM, Siddiqui MH, Ghareeb RY, et al. Maize (Zea mays L.) productivity and nitrogen use efficiency in response to nitrogen application levels and time. Front Plant Sci. 2022;13:941343.
8. Gomez KA, Gomez AA. Statistical procedures for agricultural research. 2 nd ed. Manila: International Rice Research Institute; 1983.
9. Gul H, Rahman S, Shahzad A, Gul S, Qian M, Xiao Q, et al. Maize (Zea mays L.) productivity in response to nitrogen management in Pakistan. Am J Plant Sci. 2021;12:11731179.
10. Guo Y, Xue J, Ming B, Hou J, Fan Y, Dong P, et al. Kernel hardness of different maize cultivars and the relationship between kernel hardness and moisture content. J Maize Sci. 2020;28:122-127.
11. Hammad HM, Ahmad A, Farhad W, Abbas F, Qasim K, Saeed S. Nitrogen stimulates phenological traits, growth and growing degree days of maize. Pak J Agric Sci. 2013;50(3):337-344.
12. Hammad HM, Ahmad A, Khaliq T, Farhad W, Mubeen M. Optimizing rate of nitrogen application for higher yield and quality in maize under semiarid environment. Crop Environ. 2011;2(1):38-41.
13. Hitesh S, Renu M. Effect of split doses of nitrogen and seed rate on protein content, protein fractions and yield of wheat. Indian J Agric Bi Sci. 2009;4:54-59.
14. Illes A, Bojtor C, Harsanyi E, Nagy J, Lengyel L, Szeles A. Optimizing nitrogen fertilization in maize production to improve yield and grain composition based on NDVI vegetation assessment. Agriculture. 2025;15(21):2279.
15. Jackson ML. Soil chemical analysis. New Delhi: Prentice Hall of India Pvt Ltd; 1973. p. 183-192.
16. Kaur A, Kumar M. Effect of different planting methods and nitrogen levels on the quality of kharif maize (Zea mays L.). Adv Res J Crop Improv. 2017;8(2):179-182.
17. Kumar A, Mishra J, Singh MP, Moharana RL, Kumar K, Singh V, et al. Interaction effect of nitrogen levels on leaf area index, dry matter accumulation and nitrogen uptake of wheat (Triticum aestivum L.). Afr J Bio Sci. 2024;6(15):12253-12264.
18. Lindiwe M, Erick S. Maize grain quality in response to the timing of nitrogen fertilizer application and environmental variation. Asian J Plant Sci. 2024;23:22-34.
19. Niaz A, Yaseen M, Arshad M, Ahmad R. Variable nitrogen rates and timing effect on yield, nitrogen uptake and economic feasibility of maize production. J Agric Res. 2014;52(1):77-89.
20. Oscar RV, Tollenaar M. Effect of genotype, nitrogen, plant density and row spacing on the area-per-leaf profile in maize. Agron J. 2006;98:94-101.
21. Rizwan M, Maqsood M, Rafiq M, Saeed M, Ali Z. Maize (Zea mays L.) response to split application of nitrogen. Int J Agric Biol. 2003;5(1):19-21.
22. Sharma R, Choudhary R, Jat BL. Effect of nitrogen and zinc fertilization on growth and productivity of maize. Int J Agric Sci. 2017;13(2):161-176.
23. Shrivastava S, Sidar RS, Patel S, Gurjar SK. Effect of nitrogen management on growth and yield of maize (Zea mays L.). Int J Res Agron. 2024;7(7):36-38.
24. Singh A, Uppal RS, Singh B, Kaur V. Effect of split application of nitrogen on growth and yield of maize. Int J Environ Sci. 2025;11(14):70-79.
25. Singh K, Singh AK, Singh RP, Singh V, Kumar A, Singh D. Influence of nitrogen on growth indices of different wheat varieties in late sown condition. Int J Plant Soil Sci. 2022;34(20):759-763.
26. Tsai CY, Hansel LW, Nelson OE. A colorimetric method of screening maize seeds for lysine content. Cereal Chem. 1972;49(5):572-579.
27. Ullah MI, Khakwani AA, Sadiq M, Awan I, Munir M, Ghazanfarullah. Effects of nitrogen fertilization rates on growth, quality and economic return of fodder maize (Zea mays L.). Sarhad J Agric. 2015;31(1):45-52.
28. Wieser H, Seilmeier W. The influence of nitrogen fertilization on quantities and proportions of different protein types in wheat flour. J Sci Food Agric. 1998;76:127131.
29. Yadete E, Gurmu S, Biya M. Effects of time and rate of nitrogen fertilizer application on phenology, growth and yield of maize at Jimma, Southwestern Ethiopia. World J Agric Sci Technol. 2024;2(4):185-197.
30. Zhang YM, Xue J, Zhai J, Zhang GQ, Zhang WX, Wang KR, et al. Does nitrogen application rate affect the moisture content of corn grains? J Integr Agric. 2021;20(10):26272638.