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

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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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)

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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Keyword

  • Plasma membrane H+-ATPase
  • 14-3-3 protein
  • Nitric oxide
  • Hydrogen gas
  • Adventitious rooting