CIPK-B is essential for salt stress signalling in Marchantia polymorpha.

Calcium signalling is central to many plant processes, with families of calcium decoder proteins having expanded across the green lineage and redundancy existing between decoders. The liverwort Marchantia polymorpha has fast become a new model plant, but the calcium decoders that exist in this species remain unclear. We performed phylogenetic analyses to identify the calcineurin B-like (CBL) and CBL-interacting protein kinase (CIPK) network of M. polymorpha. We analysed CBL-CIPK expression during salt stress, and determined protein-protein interactions using yeast two-hybrid and bimolecular fluorescence complementation. We also created genetic knockouts using CRISPR/Cas9. We confirm that M. polymorpha has two CIPKs and three CBLs. Both CIPKs and one CBL show pronounced salt-responsive transcriptional changes. All M. polymorpha CBL-CIPKs interact with each other in planta. Knocking out CIPK-B causes increased sensitivity to salt, suggesting that this CIPK is involved in salt signalling. We have identified CBL-CIPKs that form part of a salt tolerance pathway in M. polymorpha. Phylogeny and interaction studies imply that these CBL-CIPKs form an evolutionarily conserved salt overly sensitive pathway. Hence, salt responses may be some of the early functions of CBL-CIPK networks and increased abiotic stress tolerance required for land plant emergence.

Insights into methionine S-methylation in diverse organisms.

Dimethylsulfoniopropionate (DMSP) is an important marine anti-stress compound, with key roles in global nutrient cycling, chemotaxis and, potentially, climate regulation. Recently, diverse marine Actinobacteria, α- and γ-proteobacteria were shown to initiate DMSP synthesis via the methionine (Met) S-methyltransferase enzyme (MmtN), generating S-methyl-Met (SMM). Here we characterize a roseobacterial MmtN, providing structural and mechanistic insights into this DMSP synthesis enzyme. We propose that MmtN uses the proximity and desolvation mechanism for Met S-methylation with two adjacent MmtN monomers comprising the Met binding site. We also identify diverse functional MmtN enzymes in potentially symbiotic archaeal Candidatus Woesearchaeota and Candidate Phyla Radiation (CPR) bacteria, and the animalcule Adineta steineri, not anticipated to produce SMM and/or DMSP. These diverse MmtN enzymes, alongside the larger plant MMT enzyme with an N-terminus homologous to MmtN, likely utilize the same proximity and desolvation mechanism. This study provides important insights into the catalytic mechanism of SMM and/or DMSP production, and proposes roles for these compounds in secondary metabolite production, and SMM cycling in diverse organisms and environments.

Optogenetic control of gene expression in plants in the presence of ambient white light.

Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR-Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

Plant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well-established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron-mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small-scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron-mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron-mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.

Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice.

Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.

Calcium/Calmodulin-dependent protein kinase is negatively and positively regulated by calcium, providing a mechanism for decoding calcium responses during symbiosis signaling.

The establishment of symbiotic associations in plants requires calcium oscillations that must be decoded to invoke downstream developmental programs. In animal systems, comparable calcium oscillations are decoded by calmodulin (CaM)-dependent protein kinases, but symbiotic signaling involves a calcium/CaM-dependent protein kinase (CCaMK) that is unique to plants. CCaMK differs from the animal CaM kinases by its dual ability to bind free calcium, via calcium binding EF-hand domains on the protein, or to bind calcium complexed with CaM, via a CaM binding domain. In this study, we dissect this dual regulation of CCaMK by calcium. We find that calcium binding to the EF-hand domains promotes autophosphorylation, which negatively regulates CCaMK by stabilizing the inactive state of the protein. By contrast, calcium-dependent CaM binding overrides the effects of autophosphorylation and activates the protein. The differential calcium binding affinities of the EF-hand domains compared with those of CaM suggest that CCaMK is maintained in the inactive state at basal calcium concentrations and is activated via CaM binding during calcium oscillations. This work provides a model for decoding calcium oscillations that uses differential calcium binding affinities to create a robust molecular switch that is responsive to calcium concentrations associated with both the basal state and with oscillations.

Phosphorylation of S344 in the calmodulin-binding domain negatively affects CCaMK function during bacterial and fungal symbioses.

Calcium and Ca(2+)/calmodulin-dependent protein kinase (CCaMK) plays a critical role in the signaling pathway that establishes root nodule symbiosis and arbuscular mycorrhizal symbiosis. Calcium-dependent autophosphorylation is central to the regulation of CCaMK, and this has been shown to promote calmodulin binding. Here, we report a regulatory mechanism of Medicago truncatula CCaMK (MtCCaMK) through autophosphorylation of S344 in the calmodulin-binding/autoinhibitory domain. The phospho-ablative mutation S344A did not have significant effect on its kinase activities, and supports root nodule symbiosis and arbuscular mycorrhizal symbiosis, indicating that phosphorylation at this position is not required for establishment of symbioses. The phospho-mimic mutation S344D show drastically reduced calmodulin-stimulated substrate phosphorylation, and this coincides with a compromised interaction with calmodulin and its interacting partner, IPD3. Functional complementation tests revealed that the S344D mutation blocked root nodule symbiosis and reduced the mycorrhizal association. Furthermore, S344D was shown to suppress the spontaneous nodulation associated with a gain-of-function mutant of MtCCaMK (T271A), revealing that phosphorylation at S344 of MtCCaMK is adequate for shutting down its activity, and is epistatic over previously identified T271 autophosphorylation. These results reveal a mechanism that enables CCaMK to 'turn off' its function through autophosphorylation.

A GRAS-type transcription factor with a specific function in mycorrhizal signaling.

Legumes establish mutualistic associations with mycorrhizal fungi and with nitrogen-fixing rhizobial bacteria. These interactions occur following plant recognition of Nod factor from rhizobial bacteria and Myc factor from mycorrhizal fungi. A common symbiosis signaling pathway is involved in the recognition of both Nod factor and Myc factor and is required for the establishment of these two symbioses. The outcomes of these associations differ, and therefore, despite the commonality in signaling, there must be mechanisms that allow specificity. In Nod factor signaling, a complex of GRAS-domain transcription factors controls gene expression downstream of the symbiosis signaling pathway. Here, we show that a GRAS-domain transcription factor, RAM1, functions in mycorrhizal-specific signaling. Plants mutated in RAM1 are unable to be colonized by mycorrhizal fungi, with a defect in hyphopodia formation on the surface of the root. RAM1 is specifically required for Myc factor signaling and appears to have no role in Nod factor signaling. RAM1 regulates the expression of RAM2, a glycerol-3-phosphate acyl transferase that promotes cutin biosynthesis to enhance hyphopodia formation. We conclude that mycorrhizal signaling downstream of the symbiosis-signaling pathway has parallels with nodulation-specific signaling and functions to promote mycorrhizal colonization by regulating cutin biosynthesis.