Plant natriuretic peptide active site determination and effects on cGMP and cell volume regulation

Abstract

Natriuretic peptides (NP) were first identified in animals where they play a role in the regulation of salt and water balance. This regulation is partly mediated by intracellular changes in cyclic GMP (cGMP). NP immunoanalogues occur in many plants and have been isolated, with two NP encoding genes characterised in Arabidopsis thaliana L. (AtPNP-A and AtPNP-B). Part of AtPNP-A contains the region with homology to human atrial (A)NP. We report here on the effects of recombinant AtPNP-A and smaller synthetic peptides within the ANP-homologous region with a view to identifying the biologically active domain of the molecule. Furthermore, we investigated interactions between AtPNP-A and the hormone, abscisic acid (ABA). ABA does not significantly affect Arabidopsis mesophyll protoplast volume regulation, whereas AtPNP-A and synthetic peptides promote water uptake into the protoplasts causing swelling. This effect is promoted by the membrane permeable cGMP analogue, 8-Br-cGMP, and inhibited by guanylate cyclase inhibitors indicating that increases in cGMP are an essential component of the plant natriuretic peptides (PNP) signalling cascade. ABA does not induce cGMP transients and does not affect AtPNP-A dependent cGMP increases, hence the two regulators differ in their second messenger signatures. Interestingly, AtPNP-A significantly delays and reduces the extent of ABA stimulated stomatal closure that is also based on cell volume regulation. We conclude that a complex interplay between observed PNP effects (stomatal opening and protoplast swelling) and ABA is likely to be cell type specific.

The Phytosulfokine (PSK) Receptor Is Capable of Guanylate Cyclase Activity and Enabling Cyclic GMP-dependent Signaling in Plants

“…The levels of cGMP detected in the protoplasts are in the normal range reported for plants (e.g. (31,(37)(38)(39)(40)(41)). Importantly, cGMP levels are observed to rise in response to PSK-␣ but not the non-sulfated PSK backbone (Fig.…”

“…Synthesized phytosulfokines were used in protoplast assays. A. thaliana (Col-0) plants were grown, and leaves from 5-6-week-old plants were used to prepare protoplasts as described previously (31). Freshly isolated protoplasts (2.5 ϫ 10 5 /treatment) were used directly or in some instances pretreated for 45 min with the phosphodiesterase inhibitor dipyridamole (100 M) before a 15-min treatment with PSK-␣ or nPSK and then placed in liquid nitrogen to stop any further reactions and stored at Ϫ80°C before measuring cGMP production.…”

The multifaceted role of ABA in disease resistance

“…The ABI4 (ABSCISIC ACID INSENSITIVE 4) transcription factor [23], which integrates light-dependent ROS, sucrose and ABA signaling, acts as a positive regulator of ABA-dependent callose deposition against fungal pathogens [25]. Two closely related protein phosphatase 2C homologues, ABI1 and ABI2, function as negative regulators of the ABA pathway [11]. The activity of ABI1 and ABI2 is repressed by ABA-induced ROS [15], thereby stimulating the downstream pathway.…”

“…citri mimics a plant natriuretic peptide to antagonize ABA-dependent stomatal closure [10]. These peptides are produced in response to biotic and abiotic stress, during which they regulate cell water homeostasis and stomatal aperture through cyclic GMP-dependent signaling [11]. Fungal virulence factors, such as fusicoccin and oxalate, can also antagonize stomatal closure [12,13].…”

Plant natriuretic peptides

“…15,16 Meanwhile, the concentration and distribution of PNP may be regulated by a clearance receptor. Once the clearance receptor binds to PNP, the "ligand-receptor" complex will be internalized into the cell and disassociated.…”