Malectin-like receptor-like kinases mediate pollen tube reception in Arabidopsis thaliana

Development of plant organs requires constant monitoring of endogenous and environmental cues for which plant cells have evolved a wide array of sensors. Among them, the receptor-like kinase (RLK) family is of particular relevance for plants due to its remarkable expansion during plant evolution. RLKs sense exogenous and endogenous signals, controlling a myriad of processes such as responses to hormones and pathogens. The Catharanthus roseus RLK 1-like (CrRLK1L) group of RLKs is known to control immunity and developmental processes like reproduction and cell expansion, and have been postulated to act as signalling hubs in which several cellular pathways converge. This is best exemplified by its most studied member, FERONIA (FER), a receptor of rapid alkalinisation factor (RALF) peptides and cell wall components, and a mediator of receptor complex assembly during pathogen perception. In this project the Arabidopsis CrRLK1L receptor family was examined with the goal of identifying novel functions in plant development for uncharacterised or already characterised members. A CrRLK1L T-DNA mutant set was obtained and several higher order mutants generated to reveal defects masked by functional redundancy between related CrRLK1Ls. Initial screens identified roles for HERCULES RECEPTOR KINASE 1 (HERK1), HERK2 and THESEUS1 (THE1) in abscisic acid signalling during germination and a role during reproduction for HERK1 and its uncharacterised homolog ANJEA (ANJ). Further experiments confirmed the involvement of HERK1 and ANJ in controlling pollen tube reception in the ovule. HERK1 and ANJ were found to localise in the filiform apparatus of the synergid cells, to influence the relocalisation of NORTIA after pollen tube arrival and to physically associate with LORELEI. Additionally, the Arabidopsis RALF family was probed to identify regulators of reproduction, yielding the identification of peptides RALFL4 and RALFL19 as determinants of pollen tube growth stability

Cr RLK 1L receptor‐like kinases HERK 1 and ANJEA are female determinants of pollen tube reception

Communication between the gametophytes is vital for angiosperm fertilisation. Multiple CrRLK1L‐type receptor kinases prevent premature pollen tube burst, while another CrRLK1L protein, FERONIA (FER), is required for pollen tube reception in the female gametophyte. We report here the identification of two additional CrRLK1L homologues, HERCULES RECEPTOR KINASE 1 (HERK1) and ANJEA (ANJ), which act redundantly to promote pollen tube growth arrest at the synergid cells. HERK1 and ANJ localise to the filiform apparatus of the synergid cells in unfertilised ovules, and in herk1 anj mutants, a majority of ovules remain unfertilised due to pollen tube overgrowth, together indicating that HERK1 and ANJ act as female determinants for fertilisation. As in fer mutants, the synergid cell‐specific, endomembrane protein NORTIA (NTA) is not relocalised after pollen tube reception; however, unlike fer mutants, reactive oxygen species levels are unaffected in herk1 anj double mutants. Both ANJ and HERK1 associate with FER and its proposed co‐receptor LORELEI (LRE) in planta. Together, our data indicate that HERK1 and ANJ act with FER to mediate female–male gametophyte interactions during plant fertilisation

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase

Flavoproteinsparticipateinawidevarietyofphysiologicallyrelevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FADmoleculespermonomerinredoxcommunicationwithanactive disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein “DDOR” (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-basedtransferofreducingequivalentsinbacterialmembranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.Spanish Ministerio de Economía, Industria y Competitividad BFU2016-80343-P, BIO2016-75634-

Determinación de la estructura primaria del virus latente del clavel e identificación de regiones reguladoras en el ciclo de infección

[EN] The complete genome sequence of the Carnation latent virus (CLV) has been determined. Phylogeny analysis revealed the location of CLV within the rest of the Carlavirus genus members. Additionally, subgenomic RNA features and poly(A) tail length were studied.[ES] En este trabajo se determinó la secuencia de nucléotidos completa del genoma del Virus latente del clavel (CLV). Mediante análisis de filogenia se concretó la posición del CLV dentro del género Carlavirus. Además, se estudiaron características de los RNAs subgenómicos y la longitud de la cola poli(A).Galindo Trigo, S. (2014). Determinación de la estructura primaria del virus latente del clavel e identificación de regiones reguladoras en el ciclo de infección. http://hdl.handle.net/10251/51714Archivo delegad

Emerging mechanisms to fine-tune receptor kinase signaling specificity

Organisms need to constantly inform their cellular machinery about the biochemical and physical status of their surroundings to adapt and thrive. While some external signals are also sensed intracellularly, a considerable share of external information is registered already at the plasma membrane (PM). Receptor kinases (RKs) are crucial for plant cells to integrate such cues from the environment, from microbes, or from other cells to coordinate their physiological response and their development. Early studies on RK signaling depicted the path from external signal to internal response in a linear fashion, but recent findings show that these cellular information highways are highly interconnected and pass signals through molecular intersections. In this review, we first discuss how individual RKs simultaneously contribute to the transduction and deconvolution of a multitude of signals by controlled assembly into diverse RK complexes, exemplified by FERONIA signaling versatility. We then elaborate on how cells can exert highly localized control over the assembly, interaction and composition of such complexes in order to attain essential cellular output specificity

Dissection of the IDA promoter identifies WRKY transcription factors as abscission regulators in Arabidopsis.

Plants shed organs such as leaves, petals, or fruits through the process of abscission. Monitoring cues such as age, resource availability, and biotic and abiotic stresses allow plants to abscise organs in a timely manner. How these signals are integrated into the molecular pathways that drive abscission is largely unknown. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene is one of the main drivers of floral organ abscission in Arabidopsis and is known to transcriptionally respond to most abscission-regulating cues. By interrogating the IDA promoter in silico and in vitro, we identified transcription factors that could potentially modulate IDA expression. We probed the importance of ERF- and WRKY-binding sites for IDA expression during floral organ abscission, with WRKYs being of special relevance to mediate IDA up-regulation in response to biotic stress in tissues destined for separation. We further characterized WRKY57 as a positive regulator of IDA and IDA-like gene expression in abscission zones. Our findings highlight the promise of promoter element-targeted approaches to modulate the responsiveness of the IDA signaling pathway to harness controlled abscission timing for improved crop productivity

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase

Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.This work was supported by Spanish Ministerio de Economía, Industria y Competitividad Grants BFU2016-80343-P and BIO2016-75634-P. R.M.B. is supported by a Ramón y Cajal contract from the Spanish Ministerio de Economía, Industria y Competitividad. L.L.-M. is supported by a postdoctoral contract from Universidad de Sevilla. The research leading to these results received funding from the European Community’s Seventh Framework Program (FP7/2007–2013) under BioStruct-X Grant Agreement 7687.Peer Reviewe