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Polyamines increase nitric oxide and reactive oxygen species in guard cells of Arabidopsis thaliana during stomatal closure

Abstract

A comprehensive study which was undertaken on the effect of three polyamines (PAs) on stomatal closure was examined in relation to nitric oxide (NO) and reactive oxygen species (ROS) levels in guard cells of Arabidopsis thaliana. Three PAs—putrescine (Put), spermidine (Spd), and spermine (Spm)—induced stomatal closure, while increasing the levels of NO as well as ROS in guard cells. The roles of NO and ROS were confirmed by the reversal of closure by cPTIO (NO scavenger) and catalase (ROS scavenger). The presence of L-NAME (NOS-like enzyme inhibitor) reversed PA-induced stomatal closure, suggesting that NOS-like enzyme played a significant role in NO production during stomatal closure. The reversal of stomatal closure by diphenylene iodonium (DPI, NADPH oxidase inhibitor) or 2-bromoethylamine (BEA, copper amine oxidase inhibitor) or 1,12 diaminododecane (DADD, polyamine oxidase inhibitor) was partial. In contrast, the presence of DPI along with BEA/DADD reversed completely the closure by PAs. We conclude that both NO and ROS are essential signaling components during Put-, Spd-, and Spm-induced stomatal closure. The PA-induced ROS production is mediated by both NADPH oxidase and amine oxidase. The rise in ROS appears to be upstream of NO. Ours is the first detailed study on the role of NO and its dependence on ROS during stomatal closure by three major PAs.

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Abbreviations

ABA:

abscisic acid

Atrboh :

Arabidopsis thaliana respiratory burst oxidase homolog

BEA:

2-bromoethylamine

CM-H2DCF DA:

5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate

Col:

Columbia

cPTIO:

2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide

CuAO:

copper amine oxidase

cuao1-1 :

copper amine oxidase1

DADD:

1,12 diaminododecane

DAF-FM DA:

4-amino-5-methyl amino-2′,7′-difluorofluorescein diacetate

DMSO:

dimethyl sulfoxide

DPI:

diphenylene iodonium chloride

L-NAME:

N-nitro-L-arginine methyl ester

nia1 :

nitrate reductase1

nia2 :

nitrate reductase2

NO:

nitric oxide

NOS:

nitric oxide synthase

NR:

nitrate reductase

PA(s):

polyamine(s)

PAO:

polyamine oxidase

pao4 :

polyamine oxidase4

Put:

putrescine

ROS:

reactive oxygen species

SHAM:

salicylhydroxamic acid

Spd:

spermidine

Spm:

spermine

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Acknowledgments

This work was supported by grants (to ASR) of a JC Bose National Fellowship (No. SR/S2/JCB-06/2006) from the Department of Science and Technology and Department of Biotechnology (No. BT/PR9227/PBD/16/748/2007), both in New Delhi. SA and GG were supported by Senior Research Fellowships of University Grants Commission. We thank Mr. Prasad, technical assistant, Central Instrumentation Laboratory, for his help in using the confocal microscope. We also thank DBT-CREBB, DST-FIST, and UGC-SAP for support of infrastructure in our Department and School.

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Correspondence to Agepati S. Raghavendra.

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Handling Editor: Bhumi Nath Tripathi

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Agurla, S., Gayatri, G. & Raghavendra, A.S. Polyamines increase nitric oxide and reactive oxygen species in guard cells of Arabidopsis thaliana during stomatal closure. Protoplasma 255, 153–162 (2018). https://doi.org/10.1007/s00709-017-1139-3

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Keywords

  • Amine oxidase
  • Arabidopsis thaliana
  • Nitric oxide
  • Polyamines
  • Oxidative stress
  • Reactive oxygen species
  • Stomatal closure