Abstract
Main conclusion
NO was involved in H2-induced adventitious rooting by regulating the protein and gene expressions of PM H+-ATPase and 14-3-3. Simultaneously, the interaction of PM H+-ATPase and 14-3-3 protein was also involved in this process.
Abstract
Hydrogen gas (H2) and nitric oxide (NO) have been shown to be involved in plant growth and development. The results in this study revealed that NO was involved in H2-induced adventitious root formation. Western blot (WB) analysis showed that the protein abundances of plasma membrane H+-ATPase (PM H+-ATPase) and 14-3-3 protein were increased after H2, NO, H2 plus NO treatments, whereas their protein abundances were down regulated when NO scavenger carboxy‐2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTI O) was added. Moreover, the mRNA abundances of the HA3 and 14-3-3(7) gene as well as the activities of PM H+-ATPase (EC 3.6.1.35) and H+ pump were in full agreement with the changes of protein abundance. Phosphorylation of PM H+-ATPase and the interaction of PM H+-ATPase and 14-3-3 protein were detected by co-immunoprecipitation analysis. H2 and NO significantly up regulated the phosphorylation of PM H+-ATPase and the interaction of PM H+-ATPase and 14-3-3 protein. Conversely, the stimulation of PM H+-ATPase phosphorylation and protein interaction were significantly diminished by cPTIO. Protein interaction activator fusicoccin (FC) and inhibitor adenosine monophosphate (AMP) of PM H+-ATPase and 14-3-3 were used in this study, and the results showed that FC significantly increased the abundances of PM H+-ATPase and 14-3-3, while AMP showed opposite trends. We further proved the critical roles of PM H+-ATPase and 14-3-3 protein interaction in NO–H2-induced adventitious root formation. Taken together, our results suggested that NO might be involved in H2-induced adventitious rooting by regulating the expression and the interaction of PM H+-ATPase and 14-3-3 protein.
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Abbreviations
- H2 :
-
Hydrogen gas
- NO:
-
Nitric oxide
- NR:
-
Nitrate reductase
- WB:
-
Western blot
- PM H+-ATPase:
-
Plasma membrane H+-ATPase
- cPTIO:
-
Carboxy‐2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3-oxide
- CO-IP:
-
Co-immunoprecipitation
- FC:
-
Fusicoccin
- AMP:
-
Adenosine monophosphate
- HRW:
-
Hydrogen-rich water
- SNP:
-
Donor sodium nitroprusside
- PVP:
-
Polyvinylpolypyrrolidone
- TCA:
-
Tricarboxylic acid
- DTT:
-
Dithiothreitol
- CHAPS:
-
3-[(3-Cholamidopropyl)dimethylammo-nio]-1-propanesul-fonate
- BCA:
-
Bicinchoninic acid
- PVDF:
-
Polyvinylidene fluoride
- PMSF:
-
Phenylmethylsulfonyl fluoride
- ATP-Na2 :
-
Adenosine 5′-triphosphate disodium salt
- BTP/MES:
-
BIS–TRIS propane/morpholine ethanesulfonic acid
- SDS:
-
Sodium dodecyl sulfate
- AO:
-
Acridine orange
- POD:
-
Peroxidase
- IAAO:
-
Indoleacetic acid oxidase
- PPO:
-
Polyphenol oxidase
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Acknowledgements
This work was supported by the Discipline Construction Funds for Horticulture, Gansu Agricultural University, China (GAU-XKJS-2018-228); the National Natural Science Foundation of China (nos. 31860568, 31560563 and 31160398); the National Key Research and Development Program (2018YFD1000800); the Research Fund of Higher Education of Gansu, China (no. 2018C-14); the Post-Doctoral Foundation of China (nos. 20100470887 and 2012T50828) and the Natural Science Foundation of Gansu Province, China (nos. 1606RJZA073 and 1606RJZA077).
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425_2020_3416_MOESM1_ESM.tif
Effects of different concentrations of HRW on t adventitious rooting of cucumber. The primary root system was removed from hypocotyls of 5-day-old germinated cucumber. The number and the length of cucumber adventitious root were detected after 5-days different concentrations of HRW (a) and NO donor SNP (b) treatment. Bar = 1 cm. The values (mean ± SE) are the average of three independent experiments (n=10 explants from each of three independent experiments). Values of not sharing the same letters in a column were significantly different by Duncan’s multiple comparison test (p<0.05). Con: Control (distilled water) (TIF 26125 kb)
425_2020_3416_MOESM2_ESM.tif
Effects of different concentrations of FC and AMP on adventitious rooting of cucumber. The primary root system was removed from hypocotyls of 5-day-old germinated cucumber. The number and the length of cucumber adventitious root were detected after 5-days different concentrations of FC (a) and AMP (b) treatment. Bar = 1 cm. The values (mean ± SE) are the average of three independent experiments (n=10 explants from each of three independent experiments). Bars with different letters indicated significant differences (p<0.05) according to Duncan’s multiple range test. Con: Control (distilled water) (TIF 16860 kb)
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Li, C., Huang, D., Wang, C. et al. NO is involved in H2-induced adventitious rooting in cucumber by regulating the expression and interaction of plasma membrane H+-ATPase and 14-3-3. Planta 252, 9 (2020). https://doi.org/10.1007/s00425-020-03416-z
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DOI: https://doi.org/10.1007/s00425-020-03416-z