Since the first plant cyclic nucleotide-gated ion channel (CNGC), HvCBT1, was identified as a calmodulin binding protein, more than a decade has passed and a substantial amount of work has been done to understand the molecular nature and function of these channel proteins. Based on electrophysiological and heterologous expression analyses, plant CNGCs function as non-selective cation channels and, so far, their biological roles have been reported in defense responses, development, and ion homeostasis. Forward genetic approaches identified four AtCNGCs (AtCNGC2, 4, 11, and 12) to be involved in plant immunity, as null mutants for AtCNGC2, 4, 11, and 12 as well as a gain-of- function mutant for AtCNGC11 and 12 exhibited alterations in defense responses. Since ion flux changes have been reported as one of the early events upon pathogen recognition and also are an essential component for the activation of defense responses, the involvement of CNGCs in these ion flux changes has been suggested. However, the recent detailed characterization of null mutants suggested a more complex involvement of this channel family. In this review, we focus on the discoveries and characterization of these CNGC mutants and discuss possible roles of CNGCs as components in plant immunity.

SUMMARYThe Arabidopsis protein AtTTM3 belongs to the CYTH superfamily named after its two founding members, the CyaB adenylate cyclase from Aeromonas hydrophila and the mammalian thiamine triphosphatase. In this study we report the three-dimensional structure of a plant CYTH domain protein, AtTTM3, determined at 1.9 A resolution. The crystal structure revealed the characteristic tunnel architecture of CYTH proteins, which specialize in the binding of nucleotides and other organic phosphates and in phosphoryl transfer reactions. The b barrel is composed of eight antiparallel b strands with a cluster of conserved inwardly facing acidic and basic amino acid residues. Mutagenesis of these residues in the catalytic core led to an almost complete loss of enzymatic activity. We established that AtTTM3 is not an adenylate cyclase. Instead, the enzyme displayed weak NTP phosphatase as well as strong tripolyphosphatase activities similar to the triphosphate tunnel metalloenzyme proteins from Clostridium thermocellum (CthTTM) and Nitrosomonas europaea (NeuTTM). AtTTM3 is most highly expressed in the proximal meristematic zone of the plant root. Furthermore, an AtTTM3 T-DNA insertion knockout line displayed a delay in root growth as well as reduced length and number of lateral roots, suggesting a role for AtTTM3 in root development.

ORCID IDs: 0000-0003-3889-6183 (W.M.); 0000-0002-3797-4277 (K.Y.).The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2. Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.

The triphosphate tunnel metalloenzyme (TTM) superfamily represents a group of enzymes that is characterized by their ability to hydrolyze a range of tripolyphosphate substrates. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes, AtTTM1, AtTTM2, and AtTTM3. Although AtTTM3 has previously been reported to have tripolyphosphatase activity, recombinantly expressed AtTTM2 unexpectedly exhibited pyrophosphatase activity. AtTTM2 knockout mutant plants exhibit an enhanced hypersensitive response, elevated pathogen resistance against both virulent and avirulent pathogens, and elevated accumulation of salicylic acid (SA) upon infection. In addition, stronger systemic acquired resistance compared with wild-type plants was observed. These enhanced defense responses are dependent on SA, PHYTOALEXIN-DEFICIENT4, and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. Despite their enhanced pathogen resistance, ttm2 plants did not display constitutively active defense responses, suggesting that AtTTM2 is not a conventional negative regulator but a negative regulator of the amplification of defense responses. The transcriptional suppression of AtTTM2 by pathogen infection or treatment with SA or the systemic acquired resistance activator benzothiadiazole further supports this notion. Such transcriptional regulation is conserved among TTM2 orthologs in the crop plants soybean (Glycine max) and canola (Brassica napus), suggesting that TTM2 is involved in immunity in a wide variety of plant species. This indicates the possible usage of TTM2 knockout mutants for agricultural applications to generate pathogen-resistant crop plants.

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