P2K1 Receptor, Heterotrimeric Gα Protein and CNGC2/4 Are Involved in Extracellular ATP-Promoted Ion Influx in the Pollen of Arabidopsis thaliana

Abstract

As an apoplastic signal, extracellular ATP (eATP) is involved in plant growth and development. eATP promotes tobacco pollen germination (PG) and pollen tube growth (PTG) by stimulating Ca2+ or K+ absorption. Nevertheless, the mechanisms underlying eATP-stimulated ion uptake and their role in PG and PTG are still unclear. Here, ATP addition was found to modulate PG and PTG in 34 plant species and showed a promoting effect in most of these species. Furthermore, by using Arabidopsis thaliana as a model, the role of several signaling components involved in eATP-promoted ion (Ca2+, K+) uptake, PG, and PTG were investigated. ATP stimulated while apyrase inhibited PG and PTG. Patch-clamping results showed that ATP promoted K+ and Ca2+ influx into pollen protoplasts. In loss-of-function mutants of P2K1 (dorn1-1 and dorn1-3), heterotrimeric G protein α subunit (gpa1-1, gpa1-2), or cyclic nucleotide gated ion channel (cngc2, cngc4), eATP-stimulated PG, PTG, and ion influx were all impaired. Our results suggest that these signaling components may be involved in eATP-promoted PG and PTG by regulating Ca2+ or K+ influx in Arabidopsis pollen grains.

Dynamic Expression, Differential Regulation and Functional Diversity of the CNGC Family Genes in Cotton

“…Generally, most of the highly expressed GhCNGCs showed higher levels in reproductive tissues than vegetative tissues, implicating that GhCNGCs may be critical to cotton reproduction. For example, GhCNGC1, 13, 18, and 32 were expressed most highly in ovules (0, 1, 20 DPA), anther, filament, petal and sepal; their closest homologs are AtCNGC2 and AtCNGC4 in Arabidopsis (Figure 1), which play important functions in the regulation of floral transition [66,68], pollen growth [69], thermal sensing and acquired thermotolerance [66], senescence and programmed cell death [70][71][72], and innate immune response [65,73]. Interestingly, GhCNGC17, 33, 37, and 39 increased expression levels during the developmental stage from 10 to 25 DPA (Figure 7A), likely contributing to fiber development.…”

Damage Signaling by Extracellular Nucleotides: A Role for Cyclic Nucleotides in Elevating Cytosolic Free Calcium?

“…eATP is now reported to act upstream of CNGC2. Wu et al (2021) reported that eATP (0.1 mM) failed to elicit a plasma membrane Ca 2+ -permeable influx conductance in Arabidopsis pollen grain protoplasts of a cngc2 mutant. This concentration of eATP stimulated pollen germination in the wild type but not cngc2 ( Wu et al, 2021 ), but it remains unknown whether activation of CNGC2 by eATP would increase [Ca 2+ ] cyt .…”

“… Wu et al (2021) reported that eATP (0.1 mM) failed to elicit a plasma membrane Ca 2+ -permeable influx conductance in Arabidopsis pollen grain protoplasts of a cngc2 mutant. This concentration of eATP stimulated pollen germination in the wild type but not cngc2 ( Wu et al, 2021 ), but it remains unknown whether activation of CNGC2 by eATP would increase [Ca 2+ ] cyt . Here, the cngc2 loss of function mutant defense not death 1 ( dnd1 ; which constitutively expresses cytosolic (apo)aequorin as a luminescent [Ca 2+ ] cyt reporter, Qi et al, 2010 ) was tested for impaired eATP-induced [Ca 2+ ] cyt increase.…”

“…Annexin4 supports an eATP-induced [Ca 2+ ] cyt increase when expressed in Xenopus oocytes, but its in planta activity has not yet been reported ( Ma et al, 2019 ). Recently, a patch clamp study on Arabidopsis pollen grains identified two cyclic nucleotide-gated channel (CNGC) subunits (CNGC2 and CNGC4) as underpinning a plasma membrane eATP-activated Ca 2+ influx conductance that may be relevant to germination ( Wu et al, 2021 ). The conductance was lost in the dorn1 loss of function mutants suggesting the channels act downstream of this receptor although it was not checked whether CNGC2 and CNGC4 expression were downregulated in dorn1 .…”

“…The CNGC2 and CNGC4 channel subunits form a heterotetrameric plasma membrane Ca 2+ influx channel in flg22 pathogen-associated molecular pattern-triggered immunity (PAMP; Tian et al, 2019 ), indicating a possible common point in DAMP and PAMP signaling. Furthermore, the results of Wu et al (2021) imply that the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) or 3',5'-cyclic guanosine monophosphate (cGMP) act downstream of eATP and DORN/P2K1. Although eATP has long been known to increase animal cell cAMP ( Choi and Kim, 1997 ; Castro et al, 1998 ), there appear to be no reports to date of eATP’s having an effect on cAMP or cGMP levels in plants, but these nucleotides can increase [Ca 2+ ] cyt through CNGC activation (reviewed by Jarratt-Barnham et al, 2021 ).…”