Summary: Pattern recognition receptors (PRRs) sense microbial patterns and activate innate immunity against attempted microbial invasions. The leucine‐rich repeat receptor kinases (LRR‐RK) FLS2 and EFR, and the LRR receptor protein (LRR‐RP) receptors RLP23 and RLP42, respectively, represent prototypical members of these two prominent and closely related PRR families. We conducted a survey of Arabidopsis thaliana immune signaling mediated by these receptors to address the question of commonalities and differences between LRR‐RK and LRR‐RP signaling. Quantitative differences in timing and amplitude were observed for several early immune responses, with RP‐mediated responses typically being slower and more prolonged than those mediated by RKs. Activation of RLP23, but not FLS2, induced the production of camalexin. Transcriptomic analysis revealed that RLP23‐regulated genes represent only a fraction of those genes differentially expressed upon FLS2 activation. Several positive and negative regulators of FLS2‐signaling play similar roles in RLP23 signaling. Intriguingly, the cytoplasmic receptor kinase BIK1, a positive regulator of RK signaling, acts as a negative regulator of RP‐type immune receptors in a manner dependent on BIK1 kinase activity. Our study unveiled unexpected differences in two closely related receptor systems and reports a new negative role of BIK1 in plant immunity.

Background: The Arabidopsis response regulator 22 (ARR22) is one of two members of a recently defined novel group of two-component system (TCS) elements. TCSs are stimulus perception and response modules of prokaryotic origin, which signal by a His-to-Asp phosphorelay mechanism. In plants, TCS regulators are involved in hormone response pathways, such as those for cytokinin and ethylene. While the functions of the other TCS elements in Arabidopsis, such as histidine kinases (AHKs), histidine-containing phosphotransfer proteins (AHPs) and A-type and B-type ARRs are becoming evident, the role of ARR22 is poorly understood. Results: We present evidence that ARR22 is a preferentially cytoplasmic protein, exclusively expressed in the chalaza of developing seeds. ARR22 specifically interacts with AHP2, AHP3 and AHP5 in yeast and living plant cells. Two new loss-of-function alleles, arr22-2 and arr22-3, were isolated and characterized. With respect to their morphology and metabolite status, no significant difference in the developing seeds of the arr22 mutants was observed compared to wild type. The genetic complementation of the arr22 mutants with a genomic ARR22 fragment resulted in plants (arr22/gARR22) with a pleiotropic phenotype of different penetrance. This phenotype was not observed when the phosphorylatable Asp74 of ARR22 was changed to either a dominant-active Glu or a dominant-inactive Asn. The phenotype of the arr22/gARR22 plants was comparable to that of multiple ahk, ahp and B-type arr mutants. Conclusion: Our results favor the model that ARR22 acts as a phospho-histidine phosphatase on specific AHPs in the cytoplasm of Arabidopsis chalaza cells. The lack of any aberrant morphological and metabolite phenotype in the seeds of the arr22 mutants indicates that ARR22 is probably primarily responsible for the fine tuning of specific branches of chalaza-based TCS signalling. Even when slightly mis-expressed, ARR22 interferes with hormone homeostasis in non-chalaza tissues. Our data indicate that the chromatin status might play a crucial role in maintaining the chalaza-restricted expression of ARR22.

PII signal processor proteins are wide spread in prokaryotes and plants where they control a multitude of anabolic reactions. Efficient overproduction of metabolites requires relaxing the tight cellular control circuits. Here we demonstrate that a single point mutation in the PII signaling protein from the cyanobacterium Synechocystis sp. PCC 6803 is sufficient to unlock the arginine pathway causing over accumulation of the biopolymer cyanophycin (multi-l-arginyl-poly-l-aspartate). This product is of biotechnological interest as a source of amino acids and polyaspartic acid. This work exemplifies a novel approach of pathway engineering by designing custom-tailored PII signaling proteins. Here, the engineered Synechocystis sp. PCC6803 strain with a PII-I86N mutation over-accumulated arginine through constitutive activation of the key enzyme N-acetylglutamate kinase (NAGK). In the engineered strain BW86, in vivo NAGK activity was strongly increased and led to a more than tenfold higher arginine content than in the wild-type. As a consequence, strain BW86 accumulated up to 57 % cyanophycin per cell dry mass under the tested conditions, which is the highest yield of cyanophycin reported to date. Strain BW86 produced cyanophycin in a molecular mass range of 25 to >100 kDa; the wild-type produced the polymer in a range of 30 to >100 kDa. The high yield and high molecular mass of cyanophycin produced by strain BW86 along with the low nutrient requirements of cyanobacteria make it a promising means for the biotechnological production of cyanophycin. This study furthermore demonstrates the feasibility of metabolic pathway engineering using the PII signaling protein, which occurs in numerous bacterial species.

Proteinogenic l-amino acids (l-AAs) are essential in all kingdoms as building blocks of proteins. Their d-enantiomers are also known to fulfill important functions in microbes, fungi, and animals, but information about these molecules in plants is still sparse. Previously, it was shown that d-amino acids (d-AAs) are taken up and utilized by plants, but their ways to reduce excessive amounts of them still remained unclear. Analyses of plant d-AA content after d-Ala and d-Glu feeding opened the question if exudation of d-AAs into the rhizosphere takes place and plays a role in the reduction of d-AA content in plants. The exudation of d-Ala and d-Glu could be confirmed by amino acid analyses of growth media from plants treated with these d-AAs. Further tests revealed that other d-AAs were also secreted. Nevertheless, treatments with d-Ala and d-Glu showed that plants are still able to reduce their contents within the plant without exudation. Further exudation experiments with transport inhibitors revealed that d-AA root exudation is rather passive and comparable to the secretion of l-AAs. Altogether, these observations argued against a dominant role of exudation in the regulation of plant d-AA content, but may influence the composition of the rhizosphere.

The pepper (Capsicum annuum) resistance gene bacterial spot3 (Bs3) is transcriptionally activated by the matching Xanthomonas euvesicatoria transcription-activator–like effector (TALE) AvrBs3. AvrBs3-induced Bs3 expression triggers a rapid and local cell death reaction, the hypersensitive response (HR). Bs3 is most closely related to plant flavin monooxygenases of the YUCCA (YUC) family, which catalyze the final step in auxin biosynthesis. Targeted mutagenesis of predicted NADPH- and FAD-cofactor sites resulted in Bs3 derivatives that no longer trigger HR, thereby suggesting that the enzymatic activity of Bs3 is crucial to Bs3-triggered HR. Domain swap experiments between pepper Bs3 and Arabidopsis (Arabidopsis thaliana) YUC8 uncovered functionally exchangeable and functionally distinct regions in both proteins, which is in agreement with a model whereby Bs3 evolved from an ancestral YUC gene. Mass spectrometric measurements revealed that expression of YUCs, but not expression of Bs3, coincides with an increase in auxin levels, suggesting that Bs3 and YUCs, despite their sequence similarity, catalyze distinct enzymatic reactions. Finally, we found that expression of Bs3 coincides with increased levels of the salicylic acid and pipecolic acid, two compounds that are involved in systemic acquired resistance.

Processes in the leaf apoplast are relevant for development, cell wall rheological properties, plant nutrition, sink-source portioning, microbe-host plant-interactions or intercellular information exchange and signaling and are therefore regulated or influenced by the composition of the leaf apoplastic solute. In contrast to the traditional methods for the extraction of apoplastic solutes that are more or less destructive, we propose a new method that allows extraction of leaf apoplastic solutes (i) non-invasively and, thus, (ii) over time. Moreover, the method has (iii) a high spatial resolution that allows identification of solute-microdomains in the leaf apoplast. The method was established for Arabidopsis thaliana and Vicia faba leaves but should also be applicable to other plants species with similar leaf morphologies. It is based on the infiltration of an aqueous extraction solution into the apoplast followed by its recovery seconds later, both through the stomata. By this, the apoplast (and its solutes) of an identical leaf can be sampled on successive days with negligible symplastic contamination. A spatiotemporal mapping of leaf apoplastic ion and metabolite patterns within the identical leaf opens a window of opportunity for understanding apoplast biology. As for example, the existence of apoplastic abscisic acid gradients within a leaf in response to salinity was witnessed in this study, as was the unsuspected accumulation of kaempferol glycosides in the leaf apoplast. The presented method is relevant for plant developmental biologists, phytopathologists, plant physiologists, plant nutritionists and others that need to integrate apoplast biology into their research approaches.