The effects of different pharmacological agent treatments on ROS accumulation and functional protein activities in wheat endosperm PCD

  • Ze Lin
  • Yuanhong Qi
  • Dongcheng Liu
  • Fangfang Mao
  • Xiangyi Deng
  • Jiwei Li
  • Fangzhu Mei
  • Zhuqing ZhouEmail author
Original Article


Wheat (Triticum aestivum L.) is one of the top three food crops in the world. Studies have revealed that wheat endosperm development undergoes programmed cell death (PCD) process development that may be influenced by PCD. Waterlogging and exogenous hydrogen peroxide (H2O2) treatment exacerbates wheat endosperm PCD, whereas PCD acceleration is significantly inhibited by reactive oxygen species (ROS) scavengers. To explore the physiological mechanism of waterlogging resistance in wheat, the effects of exogenous H2O2, ascorbic acid (AsA), and cyclosporin A (CsA) treatment on ROS content, antioxidant enzyme activity, release of cytochrome c, and caspase-like protease activity in the endosperm of Huamai 8 (waterlogging-tolerant wheat cultivar) and Huamai 9 (waterlogging-sensitive wheat cultivar) were studied. The results showed that exogenous H2O2 treatment resulted in an increase in ROS content, antioxidant enzyme activity, mitochondrial membrane permeability, release of cytochrome c, and caspase-like protease activity in the endosperms of both wheat cultivars, which eventually exacerbated PCD. Compared to Huamai 8, the increase in ROS content in Huamai 9 was more significant, whereas changes in antioxidant enzyme activity, cytochrome c release, mitochondrial membrane permeability, and caspase-like protease activity were smaller. Exogenous AsA treatment leads to the content of H2O2 and catalase activity decrease, which could inhibit endosperm cell death to some extent. CsA treatment effectively inhibited the increase in H2O2 content, antioxidant enzyme activity, release of cytochrome c, and caspase-like protease activity caused by exogenous H2O2 treatment, which in turn inhibited cell death. In summary, exogenous H2O2 treatment aggravates endosperm PCD, and Huamai 9 exhibited higher ROS accumulation and a weaker antioxidant enzyme system under external stress, which may be the mechanisms underlying its sensitivity to waterlogging. CsA effectively inhibited the increase in ROS, antioxidant enzyme activity, cytochrome c release, and cell death. It is possible that in wheat endosperm, mitochondria in a similar way to animal mitochondria release cytochrome c regulating PCD.


CsA AsA H2O2 Cytochrome c ROS Programmed cell death Wheat (Triticum aestivum L.) endosperm 



Programmed cell death




Ascorbate peroxidase


Reactive oxygen species


Ascorbic acid


Cysteinyl aspartate-specific proteinase




Superoxide dismutase


Cyclosporin A


Days after flowering


Mitochondrial permeability transition pore


Polyacylamide Gel Electrophoresis



This work was supported by the National Natural Foundation of China (Grant nos. 31471428).


  1. Ahmed S, Nawata E, Hosokawa M, Domae Y, Sakuratani T (2002) Alterations in photosynthesis and some anti-oxidant enzymatic activities of mumgbean subject to waterlogging. Plant Sci 163:117–123CrossRefGoogle Scholar
  2. Ameisen CJ (2002) On the origin, evolution, and nature of programmed cell death: a timeline of four billion years. Cell Death Differ 9:367–393CrossRefPubMedGoogle Scholar
  3. Athar HUR, Khan A, Ashraf M (2008) Exogenously applied ascorbic acid alleviates salt-induced oxidative stress in wheat. Environ Exp Bot 63:224–231CrossRefGoogle Scholar
  4. Bailey-Serres J, Voesenek LACJ (2008) Flooding stress: acclimations and genetic diversity. Ann Rev Plant Biol 59:313CrossRefGoogle Scholar
  5. Bonneau L, Ge Y, Drury GE, Gallois P (2008) What happened to plant caspases? J Exp Bot 59:491–499CrossRefPubMedGoogle Scholar
  6. Chen RH, Liu W, Zhang GS, Ye JX (2010) Mitochondrial proteomic analysis of cytoplasmic male sterility line and its maintainer in wheat (Triticum aestivum L.). Agric Sci China 9:771–782CrossRefGoogle Scholar
  7. Cheng XX, Yu M, Zhang N, Zhou ZQ, Xu QT, Mei FZ, Qu LH (2016) Reactive oxygen species regulate programmed cell death progress of endosperm in winter wheat (Triticum aestivum L.) under waterlogging. Protoplasma 253:311–327CrossRefPubMedGoogle Scholar
  8. Contran N, Cerana R, Crosti P, Malerba M (2007) Cyclosporin A inhibits programmed cell death and cytochrome c release induced by fusicoccin in sycamore cells. Protoplasma 231:193–199CrossRefPubMedGoogle Scholar
  9. Dat JF, Pellinen R, Beeckman Tom, De Cotte BV, Langebartels C, Kangasjärvi J, Inzé D, Breusegem FV (2003) Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco. Plant J 33:621–632CrossRefPubMedGoogle Scholar
  10. Decaudin D, Marzo I, Brenner C, Kroemer G (1998) Mitochondria in chemotherapy-induced apoptosis: a prospective novel target of cancer therapy (review). Int J Oncol 12:141–152PubMedGoogle Scholar
  11. Elstner EF, Heupel A (1976) Formation of hydrogen peroxide by isolated cell walls from Horseradish (Armoracia lapathifolia Gilib.). Planta 130:175–180CrossRefPubMedGoogle Scholar
  12. Fan HY, Zhou ZQ, Yang CN, Jiang Z, Li JT, Cheng XX, Guo YJ (2013) Effects of waterlogging on amyloplasts and programmed cell death in endosperm cells of Triticum aestivum L. Protoplasma 250:1091–1103CrossRefPubMedGoogle Scholar
  13. Fedoroff N (2006) Redox regulatory mechanisms in cellular stress responses. Ann Bot 98:289–300CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gaffal K, Friedrichs G, El-Gammal S (2007) Ultrastructural evidence for a dual function of the phloem and programmed cell death in the floral nectary of digitalis purpurea. Ann Bot 99:593–607CrossRefPubMedPubMedCentralGoogle Scholar
  15. Gechev TS, Gadjev I, Van Breusegem F, Inzé D, Dukiandjiev S, Toneva V, Minkov I (2002) Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes. Cell Mol Life Sci 59:708–714CrossRefPubMedGoogle Scholar
  16. Ghanati F, Morita A, Yokota H (2005) Effects of aluminum on the growth of tea plant and activation of antioxidant system. Plant Soil 276:133–141CrossRefGoogle Scholar
  17. Giannopolitis CN, Ries SK (1977) Superoxide dismutases: occurrence in higher plants. Plant Physiol 59:309–314CrossRefPubMedPubMedCentralGoogle Scholar
  18. Green DR, Evan GI (2002) A matter of life and death. Cancer Cell 1:19–30CrossRefPubMedGoogle Scholar
  19. Greenberg JT, Yao N (2004) The role and regulation of programmed cell death in plant–pathogen interactions. Cell Microbiol 6:201–211CrossRefPubMedGoogle Scholar
  20. Gunawardena AH, Greenwood JS, Dengler NG (2004) Programmed cell death remodels lace plant leaf shape during development. Plant Cell 16:60–73CrossRefPubMedPubMedCentralGoogle Scholar
  21. Halestrap AP, Davidson AM (1990) Inhibition of Ca2+-induced large amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cistrans isomerase and preventing it interacting with the adenine nucleotide translocase. Biochem J 268:153–160CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hatsugai N, Kuroyanagi M, Nishimura M, Hara-Nishimura I (2006a) A cellular suicide strategy of plants: vacuole-mediated cell death. Apoptosis 11:905–911CrossRefPubMedGoogle Scholar
  23. Hatsugai N, Kuroyanagi M, Nishimura M, Hara-Nishimura I (2006b) A cellular suicide strategy of plants: vacuole-mediated cell death. Apoptosis 11:905–911CrossRefPubMedGoogle Scholar
  24. Hu D, Ma G, Wang Q, Yao J, Wang Y, Pritchard HW, Wang X (2001) Spatial and temporal nature of reactive oxygen species production and programmed cell death in elm (Ulmus pumila L.) seeds during controlled deterioration. Plant Cell Environ 35:2045–2059CrossRefGoogle Scholar
  25. Jones AM (2001) Programmed cell death in development and defense. Plant Physiol 125:94–97CrossRefPubMedPubMedCentralGoogle Scholar
  26. Khan TA, Mazid M, Mohammad F (2011) A review of ascorbic acid potentialities against oxidative stress induced in plants. J Agrobiol 28:97–111CrossRefGoogle Scholar
  27. Kobayashi H, Ikeda TM, Nagata K (2013) Spatial and temporal progress of programmed cell death in the developing starchy endosperm of rice. Planta 237:1393–1400CrossRefPubMedGoogle Scholar
  28. Kovtun Y, Chiu WL, Tena G, Sheen J (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci 97:2940–2945CrossRefPubMedGoogle Scholar
  29. Kumutha D, Ezhilmathi K, Sairam RK, Srivastava GC, Deshmukh PS, Meena RC (2009) Waterlogging induced oxidative stress and antioxidant activity in pigeonpea genotypes. Biol Plant 53:75–84CrossRefGoogle Scholar
  30. Laloi C, Apel K, Danon A (2004) Reactive oxygen signalling: the latest news. Curr Opin Plant Biol 7:323–328CrossRefPubMedPubMedCentralGoogle Scholar
  31. Lam E, Kato N, Lawton M (2001) Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411:848–853CrossRefPubMedGoogle Scholar
  32. Lin J, Wang Y, Wang G (2006) Salt stress-induced programmed cell death in tobacco protoplasts is mediated by reactive oxygen species and mitochondrial permeability transition pore status. J Plant Physiol 163:731–739CrossRefPubMedGoogle Scholar
  33. Liu X, Kim CN, Yang J, Jemmerson R, Wang X (1996) Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86:147–157CrossRefGoogle Scholar
  34. Liu Q, Qiu Y, Beta T (2010) Comparison of antioxidant activities of different colored wheat grains and analysis of phenolic compounds. J Agric Food Chem 58:9235–9241CrossRefPubMedGoogle Scholar
  35. Mueller S, Riedel HD, Stremmel W (1997) Determination of catalase activity at physiological hydrogen peroxide concentrations. Anal Biochem 245:55–60CrossRefPubMedGoogle Scholar
  36. Nasibi F, Yaghoobi MM, Kalantari KM (2011) Effect of exogenous arginine on alleviation of oxidative damage in tomato plant underwater stress. J Plant Interact 6:291–296CrossRefGoogle Scholar
  37. Noreen Z, Ashraf M (2009) Assessment of variation in antioxidative defense system in salt-treated pea (Pisum sativum L.) cultivars and its putative use as salinity tolerance markers. J Plant Physiol 166:1764–1774CrossRefPubMedGoogle Scholar
  38. Ozden M, Demirel U, Kahraman A (2009) Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2. Sci Hortic 119:163–168CrossRefGoogle Scholar
  39. Rajhi I, Yamauchi T, Takahashi H, Nishiuchi S, Shiono K, Watanabe R, Mliki A, Nagamura Y, Tsutsumi N, Nishizawa NK, Nakazono M (2011) Identification of genes expressed in maize root cortical cells during lysigenous aerenchyma formation using laser microdissection and microarray analyses. New Phytol 190:351–368CrossRefPubMedGoogle Scholar
  40. Saeidi-Sar S, Afshari H, Yaghoobi SR (2013) Effects of ascorbic acid and gibberellin A3 on alleviation of salt stress in common bean (Phaseolus vulgaris L.) seedlings. Acta Physiol Plant 35:667–677CrossRefGoogle Scholar
  41. Sairam RK, Kumutha D, Ezhilmathi K, Chinnusmy V, Meena RC (2009) Waterlogging induced oxidative stress and antioxidant enzyme activities in pigeon pea. Biol Plant (Prague) 53:493–504CrossRefGoogle Scholar
  42. Saviani EE, Orsi CH, Oliveira JFP, Pinto CAF (2002) Participation of the mitochondrial permeability transition pore in nitric oxide-induced plant cell death. FEBS Lett 510:136–140CrossRefPubMedGoogle Scholar
  43. Smirnoff N (2000) Ascorbate biosynthesis and function in photoprotection. Philos Trans R Soc Lond 355:1455–1464CrossRefGoogle Scholar
  44. Tonshin AA, Saprunova VB, Solodovnikova IM, Bakeeva LE, Yaguzhinsky LS (2003) Functional activity and ultrastructure of mitochondria isolated from myocardial apoptotic tissue. Biochemistry (Mosc) 68:875–881CrossRefGoogle Scholar
  45. Torres MA, Dangl JL (2005) Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development. Curr Opin Plant Biol 8:397–403CrossRefPubMedGoogle Scholar
  46. Tsujimoto Y, Nakagawa T, Shimizu S (2006) Mitochondrial membrane permeability transition and cell death. Biochim Biophys Acta 1757:1297–1300CrossRefPubMedGoogle Scholar
  47. Vacca RA, Valenti D, Bobba A, Merafina RS, Passarella S, Marra E (2006) Cytochrome c is released in a reactive oxygen species-dependent manner and is degraded via caspase-like proteases in tobacco Bright-Yellow 2 cells en route to heat shock-induced cell death. Plant Physiol 141:208–219CrossRefPubMedPubMedCentralGoogle Scholar
  48. Vartapetian AB, Tuzhikov AI, Chichkova NV, Taliansky M, Wolpert TJ (2011) A plant alternative to animal caspases: subtilisin-like proteases. Cell Death Differ 18:1289–1297CrossRefPubMedPubMedCentralGoogle Scholar
  49. Wang Y, Li Y, Xue H, Pritchard HW, Wang X (2015) Reactive oxygen species-provoked mitochondria-dependent cell death during ageing of elm (Ulmus pumila L.) seeds. Plant J 81:438–452CrossRefPubMedGoogle Scholar
  50. Yao N, Eisfelder BJ, Marvin J, Greenberg JT (2004) The mitochondrion-an organelle commonly involved in programmed cell death in Arabidopsis thaliana. Plant J 40:596–610CrossRefPubMedGoogle Scholar
  51. Yoshida S (2003) Molecular regulation of leaf senescence. Curr Opin Plant Biol 6:79–84CrossRefPubMedGoogle Scholar
  52. Young TE, Gallie DR (1999) Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals. Plant Mol Biol 39:915–926CrossRefPubMedGoogle Scholar
  53. Yu L, Haley S, Perret J, Harris M, Wilso J, Qian M (2002) Free radical scavenging properties of wheat extracts. J Agric Food Chem 50:1619–1624CrossRefPubMedGoogle Scholar
  54. Yu CW, Murphy TM, Lin CH (2003a) Hydrogen peroxide-induced chilling tolerance in mung beans mediated through ABA-independent glutathione accumulation. Funct Plant Biol 30:955–963CrossRefGoogle Scholar
  55. Yu L, Perret J, Harris M, Wilson J, Haley S (2003b) Antioxidant properties of bran extracts from “Akron” wheat grown at different locations. J Agric Food Chem 51:1566CrossRefPubMedGoogle Scholar
  56. Yu M, Zhou ZQ, Deng XY, Li JW, Mei FZ, Qi YH (2017) Physiological mechanism of programmed cell death aggravation and acceleration in wheat endosperm cells caused by waterlogging. Acta Physiol Plant 39:1–11CrossRefGoogle Scholar
  57. Zhang LR, Li YS, Xing D, Gao CJ (2009) Characterization of mitochondrial dynamics and subcellular localization of ROS reveal that HsfA2 alleviates oxidative damage caused by heat stress in Arabidopsis. J Exp Bot 60:2073–2091CrossRefPubMedGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2019

Authors and Affiliations

  1. 1.Laboratory of Cell Biology, College of Life Science and TechnologyHuazhong Agricultural UniversityWuhanChina
  2. 2.College of Food and Biological Science and TechnologyWuhan Institute of Design and SciencesWuhanChina
  3. 3.Division of Science and TechnologyHuazhong Agricultural UniversityWuhanChina

Personalised recommendations