Modification of The Spectrophotometric Analysis Protocol by Sims D. and Gamon to Analyze Leaf Pigment Content using Green Spinach (Amaranthus hybridus) as a Model Plant

Authors

  • Yudiansyah Yudiansyah Department of Agronomy and Horticulture, Bogor Agricultural University, Bogor 16680, West Java
  • Ismiyanti Ismiyanti Department of Agronomy and Horticulture, Bogor Agricultural University, Bogor 16680, West Java
  • Sandra Arifin Aziz Department of Agronomy and Horticulture, Bogor Agricultural University, Bogor 16680, West Java

DOI:

https://doi.org/10.29244/jtcs.11.02.147-154

Keywords:

anthocyanin, carotenoid, chlorophyll, spectrophotometry

Abstract

The analysis of pigment content in plants, particularly chlorophyll a, chlorophyll b, anthocyanin, and carotenoids, is crucial to assess their physiological performance. The spectrophotometry method by Sims and Gamon offers practicality and reliable results. This research established several variables to achieve more precise results. Green spinach leaf samples were used as a model to analyze their pigment content, determining the minimum sample area or weight, the maximum limit that does not violate Beer’s law, and the temperature during analysis. The limit of quantitation (LoQ) is the smallest quantity of analytes in a sample that still meets the criteria for accuracy and precision in testing. The research results show that using a leaf punch with a diameter of 9 mm approaches the LoQ value. To avoid measurements nearing the LoQ limit, it is advisable to use leaf punch diameters of 10- or 12-mm. Leaf punches of 22 mm are still safe to use, as there has been no deviation
from Beer’s law. Using unchilled acetone at 27°C did not show significant differences compared to using cold acetone at 7°C for the content of chlorophyll a, total chlorophyll, and carotenoids. However, for chlorophyll b and anthocyanin, despite significant differences, the chlorophyll content measured at room temperature was more practical and relatively higher compared to cold acetone, thus disproving concerns about pigment damage.

References

Ali, K.A., Noraldeen, S.S., and Yaseen, A.A. (2021). An evaluation study for chlorophyll estimation techniques. Sarhad Journal of Agriculture 37, 1458–1465.

Aprilia, D.S., Fevria, R., Vauzia, and Advinda, L. (2022). The effect of ecoenzyme spraying on the number of leaves of spinach (Amaranthus hybridus L.) cultivated hydroponically. Journal of Serambi Biologi 7, 235-238.

Burbulis, N., Blinstrubienė, A., Baltušnikienė, A., and Deveikytė, J. (2022). Foliar spraying with potassium bicarbonate reduces the negative impact of drought stress on sweet basil (Ocimum basilicum L.). Plants 11,13. https://doi.org/10.3390/plants11131716

Chen, Z., Chao, J., Wang, B., Cao, H., Wang, S., and Lin, C. (2012). Study on extraction technology of the pigment from spinach. Advanced Materials Research 518–523, 3931–3937. https://doi.org/10.4028/www.scientific.net/AMR.518-523.3931

Costa, L.M.S., Vilasboa, J., Fett-Neto, A.G., Rodrigues, N.F., Bered, F., and Margis, R. (2022). Responses to submergence and

recovery in seedlings of the rheophyte Dyckia brevifolia (Bromeliaceae). Environmental and Experimental Botany 201. https://doi.org/10.1016/j.envexpbot.2022.104984

Day, R.A., and Underwood, A.L. (1999). “Analisis Kimia Kuantitatif”. (translation by Pudjaatmaka, A.H.). pp. 392-393. Erlangga.

Dudek, G., Strzelewicz, A., Krasowska, M., Rybak, A., and Turczyn, R. (2014). A spectrophotometric method for plant pigment determination and herbs classification. Chemical Papers 68, 579–583. DOI: //doi.org/10.2478/s11696-013-0502-x

Ghaffari, H., Tadayon, M.R., Bahador, M., and Razmjoo, J. (2022). Biochemical and yield response of sugar beet to drought stress and foliar application of vermicompost tea. Plant Stress 5. DOI://doi.org/10.1016/j.stress.2022.100087

Hannachi, S., Signore, A., Adnan, M., and Mechi, L. (2022). Single and associated effects of drought and heat stresses on physiological, biochemical and antioxidant machinery of four eggplant cultivars. Plants 11,18. DOI://doi.org/10.3390/plants11182404

Hazrati, S., Tahmasebi-Sarvestani, Z., Modarres- Sanavy, S.A.M., Mokhtassi-Bidgoli, A., and Nicola, S. (2016). Effects of water stress and light intensity on chlorophyll fluorescence variables and pigments of Aloe vera L. Plant Physiology and Biochemistry 106, 141-148. http://dx.doi.org/10.1016/j.plaphy.2016.04.046.

Johnston, A., Scaggs, J., Mallory, C., Haskett, A., Warner, D., Brown, E., Hammond, K., McCormick, M. M., and McDougal, O. M. (2013). A green approach to separate spinach pigments by column chromatography. Journal of Chemical Education 90, 796 798. https://doi.org/10.1021/ed300315z.

Kong, Y., and Nemali, K. (2021). Blue and far-red light affect the area and number of individual leaves to influence vegetative growth and pigment synthesis in lettuce. Frontiers in Plant Science 12, 667407. doi: 10.3389/fpls.2021.667407.

Lichtenthaler, H.K. (1987). Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Plant Cell Membranes 148, 350-382. https://doi.org/10.1016/0076-6879(87)48036-1

Magnusson, B., and Örnemark, U. (Eds.). (2014). “Eurachem Guide: The Fitness for Purpose of Analytical Methods – A Laboratory Guide to Method Validation and Related Topics” (2nd ed.). pp 22

Palta, J.P. (1990). Leaf chlorophyll content. Remote Sensing Reviews 5, 207–213. https://doi.org/10.1080/02757259009532129

Riyanto. (2014). “Validasi & Verifikasi Metode Uji: Sesuai dengan ISO/IEC 17025 Laboratorium Pengujian dan Kalibrasi (1st ed.)”. pp. 65-78.

Deepublish.

Shah, S.H., Houborg, R., and McCabe, M.F. (2017). Response of chlorophyll, carotenoid and SPAD-502 measurement to salinity and nutrient stress in wheat (Triticum aestivum L.). Agronomy 7, 61. DOI:10.3390/agronomy7030061.

Sims, D.A., and Gamon, J.A. (2002). Relationships between leaf pigment content and spectral reflectance across a wide range of species, leaf structures, and developmental stages. Remote Sensing of Environment 81, 337-354. DOI://doi.org/10.1016/s0034-4257(02)00010-x

Song, J., Yang, J., and Jeong, B.R. (2022). Alleviation of ammonium toxicity in Salvia splendens ‘Vista Red’ with silicon supplementation. Toxics 10, 8. DOI://doi.org/10.3390/toxics10080446

Sumanta, N., Haque C.I., Nishika J., and Suprakash, R. (2014). Spectrophotometric analysis of chlorophylls and carotenoids from commonly grown fern species by using various extracting solvents. Research Journal of Chemical Sciences 4, 63–69.

Sun, H., Liu, S., Chen, K., and Li, G. (2021). Spectrophotometric determination of chlorophylls in different solvents related to the leaf traits of the main tree species in Northeast China. IOP Conference Series: Earth and Environmental Science, 836. https://doi.org/10.1088/1755-1315/836/1/012008

Zeng, J., Ping, W., Sanaeifar, A., Xu, X., Luo, W., Sha, J., Huang, Z., Huang, Y., Liu, X., Zhan, B., Zhang, H., and Li, X. (2021). Quantitative visualization of photosynthetic pigments in tea leaves based on Raman spectroscopy and calibration model transfer. Plant Methods 17,1. https://doi.org/10.1186/s13007-020-00704-3

Zhengfu, J., Xisheng, L., and Peihua, S. (2018). Effect of temperature on COD measurement by UV-Vis’s spectroscopy. ACM International Conference Proceeding Series; 2018 August 11–13, Chengdu, China; ICRCA. pp. 123–126. DOI://doi.org/10.1145/3265639.3265649

Downloads

Published

2024-06-29

How to Cite

Yudiansyah, Y., Ismiyanti, I., & Aziz, S. A. (2024). Modification of The Spectrophotometric Analysis Protocol by Sims D. and Gamon to Analyze Leaf Pigment Content using Green Spinach (Amaranthus hybridus) as a Model Plant. Journal of Tropical Crop Science, 11(02), 147–154. https://doi.org/10.29244/jtcs.11.02.147-154