Inhibition of NADPH oxidase-dependent ROS synthesis in stomatal guard cells plays an important role during endophytic colonization by Pst through stomata

Coronatine Inhibits Stomatal Closure through Guard Cell-Specific Inhibition of NADPH Oxidase-Dependent ROS Production

by Toum L., Torres P. S., Gallego S. M., Benavídes M. P., Vojnov A. A., Gudesblat G. E. (2016)

Toum L¹, Torres PS¹, Gallego SM², Benavídes MP², Vojnov AA¹, Gudesblat GE³.

1 Instituto de Ciencia y Tecnología Dr. César Milstein, Fundación Pablo Cassará, Consejo Nacional de Investigaciones Científicas y Técnicas Buenos Aires, Argentina.

2 Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires Buenos Aires, Argentina.

3 Instituto de Ciencia y Tecnología Dr. César Milstein, Fundación Pablo Cassará, Consejo Nacional de Investigaciones Científicas y TécnicasBuenos Aires, Argentina; Instituto de Biodiversidad y Biología Experimental y Aplicada, Departamento de Biodiversidad y Biología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos AiresBuenos Aires, Argentina.

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In Front Plant Sci. 7: 1851 – doi: 10.3389/fpls.2016.01851 – eCollection 2016 –

https://www.ncbi.nlm.nih.gov/pubmed/28018388

Abstract

Microbes trigger stomatal closure through microbe-associated molecular patterns (MAMPs). The bacterial pathogen Pseudomonas syringae pv. tomato (Pst) synthesizes the polyketide toxin coronatine, which inhibits stomatal closure by MAMPs and by the hormone abscisic acid (ABA). The mechanism by which coronatine, a jasmonic acid-isoleucine analog, achieves this effect is not completely clear. Reactive oxygen species (ROS) are essential second messengers in stomatal immunity, therefore we investigated the possible effect of coronatine on their production. We found that coronatine inhibits NADPH oxidase-dependent ROS production induced by ABA, and by the flagellin-derived peptide flg22. This toxin also inhibited NADPH oxidase-dependent stomatal closure induced by darkness, however, it failed to prevent stomatal closure by exogenously applied H₂O₂ or by salicylic acid, which induces ROS production through peroxidases. Contrary to what was observed on stomata, coronatine did not affect the oxidative burst induced by flg22 in leaf disks. Additionally, we observed that in NADPH oxidase mutants atrbohd and atrbohd/f, as well as in guard cell ABA responsive but flg22 insensitive mutants mpk3, mpk6, npr1-3, and lecrk-VI.2-1, the inhibition of ABA stomatal responses by both coronatine and the NADPH oxidase inhibitor diphenylene iodonium was markedly reduced. Interestingly, coronatine still impaired ABA-induced ROS synthesis in mpk3, mpk6, npr1-3, and lecrk-VI.2-1, suggesting a possible feedback regulation of ROS on other guard cell ABA signaling elements in these mutants. Altogether our results show that inhibition of NADPH oxidase-dependent ROS synthesis in guard cells plays an important role during endophytic colonization by Pst through stomata.

Phytopathogens and stomatal response

 

Xanthomonas campestris overcomes Arabidopsis stomatal innate immunity through a DSF cell-to-cell signal-regulated virulence factor.

by Gudesblat G. E., Torres P. S.,

Vojnov A. A.

(2009)

in Plant Physiol. 149, 1017–1027. doi: 10.1104/pp.108.126870 –

PubMed Abstract | CrossRef Full Text | Google Scholar

http://www.plantphysiol.org/content/149/2/1017

Abstract

Pathogen-induced stomatal closure is part of the plant innate immune response. Phytopathogens using stomata as a way of entry into the leaf must avoid the stomatal response of the host.

In this article, we describe a factor secreted by the bacterial phytopathogen Xanthomonas campestris pv campestris (Xcc) capable of interfering with stomatal closure induced by bacteria or abscisic acid (ABA).

We found that living Xcc, as well as ethyl acetate extracts from Xcc culture supernatants, are capable of reverting stomatal closure induced by bacteria, lipopolysaccharide, or ABA. Xcc ethyl acetate extracts also complemented the infectivity of Pseudomonas syringae pv tomato (Pst) mutants deficient in the production of the coronatine toxin, which is required to overcome stomatal defense.

By contrast, the rpfF and rpfC mutant strains of Xcc, which are unable to respectively synthesize or perceive a diffusible molecule involved in bacterial cell-to-cell signaling, were incapable of reverting stomatal closure, indicating that suppression of stomatal response by Xcc requires an intact rpf/diffusible signal factor system.

In addition, we found that guard cell-specific Arabidopsis (Arabidopsis thaliana) Mitogen-Activated Protein Kinase3 (MPK3) antisense mutants were unresponsive to bacteria or lipopolysaccharide in promotion of stomatal closure, and also more sensitive to Pstcoronatine-deficient mutants, showing that MPK3 is required for stomatal immune response.

Additionally, we found that, unlike in wild-type Arabidopsis, ABA-induced stomatal closure in MPK3 antisense mutants is not affected by Xcc or by extracts from Xccculture supernatants, suggesting that the Xcc factor might target some signaling component in the same pathway as MPK3.

The important role of MPK3 in the perception of ABA and H2O2 in stomata

 

Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide.

by Gudesblat G. E.,

Iusem N. D., 

Morris P. C.

(2007)

in New Physiol., 173: 713-721 – doi: 10.1111/j.1469-8137.2006.01953.x –

PubMed Abstract | CrossRef Full Text | Google Scholar 

http://onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.2006.01953.x/abstract

Summary

• • MAP kinases have been linked to guard cell signalling. Arabidopsis thaliana MAP Kinase 3 (MPK3) is known to be activated by abscisic acid (ABA) and hydrogen peroxide (H₂O₂), which also control stomatal movements.
• • We therefore studied the possible role of MPK3 in guard cell signalling through guard cell-specific antisense inhibition of MPK3 expression.
• • Such transgenic plants contained reduced levels of MPK3 mRNA in the guard cells and displayed partial insensitivity to ABA in inhibition of stomatal opening, but responded normally to this hormone in stomatal closure. However, ABA-induced stomatal closure was reduced compared with controls when cytoplasmic alkalinization was prevented with sodium butyrate. MPK3 antisense plants were less sensitive to exogenous H₂O₂, both in inhibition of stomatal opening and in promotion of stomatal closure, thus MPK3 is required for the signalling of this compound. ABA-induced H₂O₂ synthesis was normal in these plants, indicating that MPK3 probably acts in signalling downstream of H₂O₂.
• • These results provide clear evidence for the important role of MPK3 in the perception of ABA and H₂O₂ in guard cells.

More brassinosteroids dramatically increase the number of stomata

Photo credit: Google

Plants make big decisions with microscopic cellular competition .

www.washington.edu1024 × 1024

A picture of stomata.

SPEECHLESS integrates brassinosteroid and stomata signalling pathways

by Gudesblat G. E., Schneider-Pizon J., Betti C., Mayerhofer J., Vanhoutte I., van Dongen W., Boeren S., Zhiponova M., de Vries S., Jonak C., Russinova E. (2012)

in Nat Cell Biol 2012,14:548-554. (PubMed Abstract | Publisher Full Text)

EXCERPT

Gent researchers at VIB have unraveled the action mechanism of the main plant hormone that regulates the development of stomata. This breakthrough has important implications for environmental research and for the protection of plants against disease and stress.

Plants breathe through stomata

Plant leaves are protected from drying out by an airtight wax layer. They breathe and release water through microscopic pores called stomata. Every year 40% of atmospheric CO₂ and twice the volume of water found in our atmosphere pass through these pores. This means that stomata are not only important for plant development but also for our climate!

It’s no surprise then that these pores appear to be strictly regulated by plants. Stomata react extremely fast to internal plant signals and changes in the environment. When rain is scarce, for example, the pores will close to prevent the plant from wasting water while an automatic drought protection mechanism is triggered into action. Brassinosteroids, a class of plant hormones, play an important role in determining the number of leaf stomata, but the underlying mechanism was until now not well understood.

Brassinosteroids are crucial plant hormones

Controlling multiple aspects of plant growth and development, brassinosteroids are omnipresent in the plant kingdom. The hormones have a positive effect on the quality and productivity of crops and increase their resistance to stress and disease.

Scientist Jenny Russinova and her team, who are associated with both VIB and Ghent University, study the action mechanisms of brassinosteroids. A recent breakthrough led them to conclude that the latter also affect the number of stomata. Plants without the hormone develop many fewer stomata. The opposite is also true: more brassinosteroids dramatically increase the number of pores.

Scientific breakthrough: action mechanism deciphered

The VIB scientists are the first to unravel the action mechanism. They were able to determine how the various agents work together to form new stomata. Their experiments showed that brassinosteroids exert direct action on speechless, the transcription factor that initiates the development of stomata. Their action allows for a multitude of different interactions. This exemplifies the strictly orchestrated regulation of stomata development, which is able to react very quickly to environmental changes or internal plant signals.

See the text: Science Daily