Evolution of the DUF26-containing proteins in plants

For plants as sessile organisms effective signaling mechanisms are essential. Plants utilize signaling networks to receive cues from the environment and signal between cells. Various proteins and protein families are involved in the signaling networks in plants including receptor-like kinases (RLKs) and their related receptor-like proteins (RLPs). RLKs are typically located in the plasma membrane and transfer signals from the apoplastic space to the interior of the cell. The domain of unknown function 26 (DUF26) is a cysteine-rich protein domain involved in signaling. DUF26-containing proteins are a plant-specific protein family containing both RLKs and RLPs, including cysteine-rich receptor-like kinases (CRKs), plasmodesmata-localized proteins (PDLPs) and cysteine-rich receptor-like secreted proteins (CRRSPs). To facilitate investigation of the functions of DUF26 proteins, comprehensive phylogenetic and evolutionary analyses were combined with broad phenotypic analyses of crk mutants and structural investigation of two PDLPs from the model species Arabidopsis thaliana. These analyses revealed that DUF26-containing genes have a complex evolutionary history, including several steps of domain rearrangements and differential expansion and contraction patterns in different groups of plants and between different groups of CRKs, PDLPs and CRRSPs. CRKs were found to be involved in stress responses and development based on their loss-of-function phenotypes. The crystal structure of the AtPDLPs revealed a close structural homology between the DUF26 domain and fungal lectins, suggesting that DUF26 could be a carbohydrate-binding unit in plants. Annotation quality is crucial for virtually any type of sequence-based analysis, including phylogenetic estimation of relationships between genes, proteins and species. For this reason, the annotations of DUF26-containing genes were carefully curated in such a way as to facilitate the subsequent evolutionary analyses. Since most functional data is obtained from model species, only through thorough estimation of the relationships between proteins from different species we can reliably transfer information among species. In the future, as more functional information becomes available, the knowledge gained from this study will be applied in translational research between model species and crop species.Kasvit tarvitsevat tehokasta viestintään solujen välillä voidakseen reagoida muutoksiin ja stressitekijöihin kasvuympäristössään. Kasvit hyödyntävät tähän tarkoitukseen muun muassa erilaisia proteiineja, yhtenä esimerkkinä reseptorinkaltaiset kinaasit (RLK:t) ja niille sukua olevat reseptorinkaltaiset proteiinit (RLP:t). RLK:t sijaitsevat yleensä kasvin solukalvolla ja siirtävät viestejä solukalvon ulkopuolelta solun sisälle. RLK-proteiineja koodaavat geenit kuuluvat useisiin geeniperheisiin. Geeniperheet ovat muodostuneet geenien monistuessa genomissa ja niiden jäsenet ovat samaa alkuperää ja yleensä sekvensseiltään samankaltaisia. Yksi sekä RLK- että RLP-proteiineja koodaava geeniperhe on DUF26-domeenin sisältävä geeniperhe, jonka evoluutiota on tutkittu tässä väitöskirjassa. DUF26-geenejä esiintyy vain kasveilla. Niiden evoluution selvittämiseksi geenit tunnistettiin kasvien genomeista ja niitä kuvaavat geenimallit tarkistettiin ja tarvittaessa korjattiin, koska geenimallien laatu on keskeistä luotettavien tulosten saamiseksi geenien sekvensseihin perustuvissa analyyseissa. DUF26-geenien sukulaisuutta selvitettiin fylogeneettisten analyysien avulla ja niiden lisäksi tutkittiin myös näiden geenien koodaamien proteiinien toimintaa lituruoholla. Lisäksi kahden DUF26-proteiinin rakenne selvitettiin kiteyttämällä. DUF26-geenien evoluutio on sisältänyt paljon muutoksia ja geeniperheen eri alaryhmät ovat kehittyneet eri tavoin. Muutoksia on tapahtunut sekä geenien rakenteessa että geenikopioiden määrässä. DUF26-geenien koodaamien RLK-proteiinien toiminta liittyy tutkimuksen perusteella viestintään stressitilanteissa. Kiderakenteen samankaltaisuus sienten hiilihydraatteja sitovien proteiinien kanssa tarkoittaa, että myös DUF26-proteiinit pystyvät mahdollisesti sitomaan hiilihydraatteja. Lisätutkimus on kuitenkin tarpeen DUF26-domeenin tarkemman biokemiallisen toiminnan ymmärtämiseksi. Proteiinien toimintaa tutkitaan yleensä mallilajeilla, kasvien tapauksessa eniten lituruoholla. Geeniperheiden, kuten DUF26-geenien, evoluution tutkiminen eri lajeilla mahdollistaa toisiaan vastaavien geenien tunnistamisen lajien välillä ja siten lituruohon avulla saadun proteiinien toimintaan liittyvän tiedon hyödyntämisen tulevaisuudessa myös viljely- ja hyötykasvien jalostuksessa kehitettäessä paremmin ympäristön stressitekijöitä kestäviä lajikkeita

High-throughput sequencing data and the impact of plant gene annotation quality

The use of draft genomes of different species and re-sequencing of accessions and populations are now a common tool for plant biology research. The de novo assembled draft genomes make it possible to identify pivotal divergence points in the plant lineage and provide an opportunity to investigate the genomic basis and timing of biological innovations by inferring orthologs between species. Furthermore, re-sequencing facilitates the mapping and subsequent molecular characterization of causative loci for traits including plant stress tolerance or development. In both cases high quality gene annotation, the identification of protein-coding regions, gene promoters and 5’ and 3’ untranslated regions, is critical for investigation of gene function. Annotations are constantly improving but automated gene annotations still require manual curation and experimental validation. This is particularly important for genes with large introns, genes located in regions rich with transposable elements or repeats, large gene families and segmentally duplicated genes. In this opinion paper we highlight the impact of annotation quality on evolutionary analyses, genome-wide association studies and the identification of orthologous genes in plants. Furthermore, we predict that incorporating the accurate information from manual curation into databases will dramatically improve the performance of automated gene predictors.Peer reviewe

Large-scale phenomics identifies primary and fine-tuning roles for CRKs in responses related to oxidative stress

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.Peer reviewe

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.Peer reviewe

Author Correction: Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch.

In the version of this article initially published, there was a mistake in the calculation of the nucleotide mutation rate per site per generation: 1 × 10−9 mutations per site per generation was used, whereas 9.5 × 10−9 was correct. This error affects the interpretation of population-size changes over time and their possible correspondence with known geological events, as shown in the original Fig. 4 and supporting discussion in the text, as well as details in the Supplementary Note. Neither the data themselves nor any other results are affected. Figure 4 has been revised accordingly. Images of the original and corrected figure panels are shown in the correction notice

Mechanistic insights into the evolution of DUF26-containing proteins in land plants

Large protein families are a prominent feature of plant genomes and their size variation is a key element for adaptation. However, gene and genome duplications pose difficulties for functional characterization and translational research. Here we infer the evolutionary history of the DOMAIN OF UNKNOWN FUNCTION (DUF) 26-containing proteins. The DUF26 emerged in secreted proteins. Domain duplications and rearrangements led to the appearance of CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASES (CRKs) and PLASMODESMATA-LOCALIZED PROTEINS (PDLPs). The DUF26 is land plant-specific but structural analyses of PDLP ectodomains revealed strong similarity to fungal lectins and thus may constitute a group of plant carbohydrate-binding proteins. CRKs expanded through tandem duplications and preferential retention of duplicates following whole genome duplications, whereas PDLPs evolved according to the dosage balance hypothesis. We propose that new gene families mainly expand through small-scale duplications, while fractionation and genetic drift after whole genome multiplications drive families towards dosage balance.Peer reviewe

CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase (RLK)-dependent signaling networks. Here we show that the cysteine-rich RLK CRK2 kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RBOHD in Arabidopsis. Functional CRK2 is required for the full elicitor-induced ROS burst and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment and substitution of S703 with alanine reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating MAMP-triggered ROS production.Peer reviewe

Integrated cluster analysis of <i>crk</i> mutant phenotypes.

<p>An age-matched collection of T-DNA insertion lines in <i>CRK</i> genes was analyzed for developmental and stress-related phenotypes. <b>(A)</b> Analysis of developmental phenotypes of <i>crk</i> mutant lines: senescence, germination (endosperm rupture), epidermal cell segmentation, bolting, flowering, and root length. <b>(B)</b> Analysis of abiotic stresses phenotypes of <i>crk</i> lines: germination of <i>crk</i> lines on medium containing NaCl, cell death (measured by electrolyte leakage) in response to Xanthine-Xanthine Oxidase (X+XO), ultraviolet light (UV-AB), ozone (O₃), or light stress. <b>(C)</b> Analysis of photosynthesis responses upon treatment with DCMU or methyl viologen (MV). <b>(D)</b> Pathogen phenotypes. ROS production in response to treatment with the bacterial elicitor flagellin (flg22). Stomatal aperture ratio in response to flg22 and chitin treatments and <i>crk</i> susceptibility to the hemibiotrophic bacterial pathogen <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 (<i>Pto</i> infection) or the biotrophic fungal pathogens <i>Golovinomyces orontii</i> (<i>Go</i>) (virulent on <i>Arabidopsis</i>) or <i>Blumeria graminis</i> f.sp. <i>hordei</i> (<i>Bgh</i>, a barley pathogen, non-pathogenic on <i>Arabidopsis</i>). <b>(E)</b> Analysis of stomatal parameters: fresh weight (for determination of water loss), density, length, aperture, stomatal aperture in response to ABA treatment, steady state stomatal conductance, stomatal closure in response to elevated CO₂, O₃, and darkness. Experiments were made comparable by bootstrap sampling to <i>n</i> = 15 followed by averaging over bootstrap estimates. Red and blue indicate statistically significant increase or decrease in response compared to Col-0 wild type, respectively, while white indicates a response that is similar to wild type Col-0. The intensity of color is proportional to the Benjamini-Hochberg false discovery rate (FDR) adjusted Z statistic which takes the estimated means and their variation into account. As a rough guideline, |Z|>1.67 corresponds to a FDR<10% (shown with light hue), and |Z|<2.6 to a strong FDR<1% (intense color). White: non-significant response; grey: not measured. A corresponding plot displaying the adjusted Z statistics without thresholding is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s025" target="_blank">S25 Fig</a>.</p