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Nitric oxide production occurs downstream of reactive oxygen species in guard cells during stomatal closure induced by chitosan in abaxial epidermis of Pisum sativum

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Abstract

The effects of chitosan (β-1,4 linked glucosamine, a fungal elicitor), on the patterns of stomatal movement and signaling components were studied. cPTIO (NO scavenger), sodium tungstate (nitrate reductase inhibitor) or l-NAME (NO synthase inhibitor) restricted the chitosan induced stomatal closure, demonstrating that NO is an essential factor. Similarly, catalase (H2O2 scavenger) or DPI [NAD(P)H oxidase inhibitor] and BAPTA-AM or BAPTA (calcium chelators) prevented chitosan induced stomatal closure, suggesting that reactive oxygen species (ROS) and calcium were involved during such response. Monitoring the NO and ROS production in guard cells by fluorescent probes (DAF-2DA and H2DCFDA) indicated that on exposure to chitosan, the levels of NO rose after only 10 min, while those of ROS increased already by 5 min. cPTIO or sodium tungstate or l-NAME prevented the rise in NO levels but did not restrict the ROS production. In contrast, catalase or DPI restricted the chitosan-induced production of both ROS and NO in guard cells. The calcium chelators, BAPTA-AM or BAPTA, did not have a significant effect on the chitosan induced rise in NO or ROS. We propose that the production of NO is an important signaling component and participates downstream of ROS production. The effects of chitosan strike a marked similarity with those of ABA or MJ on guard cells and indicate the convergence of their signal transduction pathways leading to stomatal closure.

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Abbreviations

ABA:

Abscisic acid

BAPTA:

1,2-bis(o-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

BAPTA-AM:

1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester

cPTIO:

2-Phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide

DAF-2DA:

4,5-Diaminofluorescein diacetate

DPI:

Diphenyleneiodonium chloride

H2DCFDA:

2′,7′-Dichlorodihydrofluorescein diacetate

l-NAME:

N-nitro-l-Arg-methyl ester

MES:

2-(N-morpholino) ethanesulphonic acid

MJ:

Methyl jasmonate

NO:

Nitric oxide

NOS:

Nitric oxide synthase

NR:

Nitrate reductase

ROS:

Reactive oxygen species

SNP:

Sodium nitroprusside

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Acknowledgments

This work was supported by grants from Council of Scientific and Industrial Research [No. 38(0259)/08/EMR-II], Department of Biotechnology (BT/PR9227/PBD/16/748/2007) and a JC Bose National Fellowship from Department of Science and Technology (No. SR/S2/JCB-06/2006) to A. S. Raghavendra, all from New Delhi. V. K. Gonugunta and M. R. Puli are supported by CSIR Research Fellowships, New Delhi. We also acknowledge the support from the grants of Department of Science and Technology-Fund for Improvement of Science & Technology Infrastructure (DST-FIST) and University Grants Commission-Special Assistance Program (UGC-SAP) to Department of Plant Sciences.

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  1. Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, 500046, India

    Nupur Srivastava, Vijay K. Gonugunta, Mallikarjuna R. Puli & Agepati S. Raghavendra

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  1. Nupur Srivastava
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  2. Vijay K. Gonugunta
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  3. Mallikarjuna R. Puli
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  4. Agepati S. Raghavendra
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Correspondence to Agepati S. Raghavendra.

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Nupur Srivastava and Vijay K. Gonugunta have contributed equally.

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Srivastava, N., Gonugunta, V.K., Puli, M.R. et al. Nitric oxide production occurs downstream of reactive oxygen species in guard cells during stomatal closure induced by chitosan in abaxial epidermis of Pisum sativum . Planta 229, 757–765 (2009). https://doi.org/10.1007/s00425-008-0855-5

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