Plant cyclic nucleotide-gated channels (CNGCs) are tetrameric cation channels which may be activated by the cyclic nucleotides (cNMPs) adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The genome of Arabidopsis thaliana encodes 20 CNGC subunits associated with aspects of development, stress response and immunity. Recently, it has been demonstrated that CNGC subunits form heterotetrameric complexes which behave differently from the homotetramers produced by their constituent subunits. These findings have widespread implications for future signalling research and may help explain how specificity can be achieved by CNGCs that are known to act in disparate pathways. Regulation of complex formation may involve cyclic nucleotide-gated channel-like proteins.

Mutations are the source of both genetic diversity and mutational load. However, the effects of increasing environmental temperature on plant mutation rates and relative impact on specific mutational classes (e.g., insertion/deletion [indel] vs. single nucleotide variant [SNV]) are unknown. This topic is important because of the poorly defined effects of anthropogenic global temperature rise on biological systems. Here, we show the impact of temperature increase on Arabidopsis thaliana mutation, studying whole genome profiles of mutation accumulation (MA) lineages grown for 11 successive generations at 29°C. Whereas growth of A. thaliana at standard temperature (ST; 23°C) is associated with a mutation rate of 7 × 10−9 base substitutions per site per generation, growth at stressful high temperature (HT; 29°C) is highly mutagenic, increasing the mutation rate to 12 × 10−9. SNV frequency is approximately two- to threefold higher at HT than at ST, and HT-growth causes an ∼19- to 23-fold increase in indel frequency, resulting in a disproportionate increase in indels (vs. SNVs). Most HT-induced indels are 1–2 bp in size and particularly affect homopolymeric or dinucleotide A or T stretch regions of the genome. HT-induced indels occur disproportionately in nucleosome-free regions, suggesting that much HT-induced mutational damage occurs during cell-cycle phases when genomic DNA is packaged into nucleosomes. We conclude that stressful experimental temperature increases accelerate plant mutation rates and particularly accelerate the rate of indel mutation. Increasing environmental temperatures are thus likely to have significant mutagenic consequences for plants growing in the wild and may, in particular, add detrimentally to mutational load.

Extracellular ATP (eATP) has long been established in animals as an important signalling molecule but this is less understood in plants. The identification of Arabidopsis thaliana DORN1 (Does Not Respond to Nucleotides) as the first plant eATP receptor has shown that it is fundamental to the elevation of cytosolic free Ca2+ ([Ca2+]cyt) as a possible second messenger. eATP causes other downstream responses such as increase in reactive oxygen species (ROS) and nitric oxide, plus changes in gene expression. The plasma membrane Ca2+ influx channels involved in eATP-induced [Ca2+]cyt increase remain unknown at the genetic level. Arabidopsis thaliana Annexin 1 has been found to mediate ROS-activated Ca2+ influx in root epidermis, consistent with its operating as a transport pathway. In this study, the loss of function Annexin 1 mutant was found to have impaired [Ca2+]cyt elevation in roots in response to eATP or eADP. Additionally, this annexin was implicated in modulating eATP-induced intracellular ROS accumulation in roots as well as expression of eATP-responsive genes.

Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca 2+ ] cyt ) increase as a second messenger. The downstream PM Ca 2+ channels remain enigmatic. Here, the Arabidopsis thaliana Ca 2+ channel subunit CYCLIC NUCLEOTIDE-GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca 2+ ] cyt signalling in roots.Extracellular ATP-induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca 2+ ] cyt were measured with aequorin, and root transcriptional changes were determined by quantitative real-time PCR. Two cngc2 loss-offunction mutants were used: cngc2-3 and defence not death1 (which expresses cytosolic aequorin).Extracellular ATP-induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca 2+ dependent, requiring CNGC2 but not CNGC4 (its channel cosubunit in immunity signalling). Activation of PM Ca 2+ influx currents also required CNGC2. The eATP-induced [Ca 2+ ] cyt increase and transcriptional response in cngc2 roots were significantly impaired.CYCLIC NUCLEOTIDE-GATED CHANNEL2 is required for eATP-induced epidermal Ca 2+ influx, causing depolarization leading to [Ca 2+ ] cyt increase and damage-related transcriptional response.